Diagnostic method for diseases by screening for hepcidin in human or animal tissues, blood or body fluids and therapeutic uses therefor

ABSTRACT

The present invention concerns methods and kits for diagnosing a disease condition characterized by non-physiological levels of hepcidin protein, including prohepcidin and fragments thereof, comprising obtaining a tissue or fluid sample from a subject; contacting the sample with an antibody or fragment thereof that specifically binds to a polypeptide corresponding to the mid-portion or C terminus of a hepcidin protein, and quantifying the hepcidin level using an assay based on binding of the antibody and the polypeptide; wherein the non-physiological level of hepcidin is indicative of the disease condition. The present invention also concerns diagnostic methods and kits for applications in genetic technological approaches, such as for overexpressing or down-regulating hepcidin. The present invention further concerns therapeutic treatment of certain diseases by treatment of subjects with hepcidin and agonists or antagonists of hepcidin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefits under 35 U.S.C. §371 of PCTInternational Application No. PCT/US2003/036946 filed Nov. 19, 2003,which is a Continuation-In-Part of application Ser. No. 10/441,089 filedMay 19, 2003, now U.S. Pat. No. 7,320,894, and which is aContinuation-In-Part of application Ser. No. 10/299,486 filed Nov. 19,2002, now U.S. Pat. No. 7,411,048.

BACKGROUND ART

Iron is an essential trace element that is required for growth anddevelopment of all living organisms; it is indispensable for DNAsynthesis and a broad range of metabolic processes. However,disturbances of iron metabolism have been implicated in a number ofsignificant mammalian diseases, including, but not limited to irondeficiency anemia, hemosiderosis or the iron overload diseasehemochrornatosis (Pietrangelo, A. (2002) Am J Physiol. Gastrointest.Liver Physiol. 282, G403-414; Andrews, N. C. (2000) Annu. Rev. GenomicsHum. Genet. 1, 75-98; Philpott, C. C. (2002) Hepatology 35, 993-1001;Anderson and Powell (2002) Int J Hematol 76, 203-203; Beutler et al.,(2001) Drug-Metab. Dispos. 29, 495-499). Under physiological conditions,a human's iron content is regulated by controlling absorption. Inmammals, iron absorption occurs predominantly in the duodenum and upperjejunum, and is the only mechanism by which iron stores arephysiologically controlled (Philpott, C. C. (2002) Hepatology 35,993-1001). Following absorption, iron is bound to circulatingtransferrin and delivered to tissues throughout the body. In the liver,the major site of iron storage, transferrin-bound iron is taken into thecells by receptor-mediated endocytosis via the classical transferrinreceptor (TfR1) (Collawn et al. (1990) Cell 63, 1061-1072) andpresumably in greater amounts via the recently identified homologoustransferrin receptor 2 (TfR2) (Kawabata et al. (1999) J Biol Chem 274,20826-20832). The extracellular domain of this protein is 45% identicalto the corresponding portion of TfR1 (Id.). TfR2 can also bind diferrictransferrin and facilitate the uptake of iron. Mutations in TfR2 havebeen associated with certain forms of hemochromatosis demonstrating theimportant role for TfR2 in iron homeostasis (Philpott, C. C. (2002)Hepatology 35, 993-1001; Camasehella et al., (2000) Nat. Genet. 25,14-15; Fleming et al., (2002) Proc. Natl. Acad. Sci. USA 99,10653-10658). TfR2 is predominantly expressed in the liver (Fleming etal., (2000) Proc. Natl. Acadi. Sci. USA 97, 2214-2219; Subramaniam etal., 2002) Cell Biochem. Biophys. 36, 235-239), however, the exactcellular localization is still unknown.

A feedback mechanism exists that enhances iron absorption in individualswho are iron deficient, whereas iron absorption is reduced in personswith iron overload (Pietrangelo, A. (2002) Am J Physiol GastrointestLiver Physiol 282, G403-414; Philpott, C. C. (2002) Hepatology 35,993-1001; Anderson and Powell (2002) Int J Hematol 76, 203-203). Inhereditary hemochromatosis (HH), however, this regulatory mechanismseems to be impaired; despite iron overload, elevated amounts of ironare absorbed from the diet and lead to accumulation of excess iron ininternal organs, resulting in organ dysfunction and failure. Themolecular mechanisms by which the intestine responds to alterations inbody iron requirements is poorly understood. In this context, hepcidin,a recently identified mammalian peptide (Krause et al. (2000) FEBS Lett489, 147-150; Park et al. (2001) J Biol Chem 276, 7806-7810), ispredicted as a key signaling component regulating iron homeostasis(Philpott C. C. (2002) Hepatology 35, 993-1001; Nicolas et al. (2002)Proc Natl Acad Sci USA 99, 4596-4601)

Hepcidin is a small cysteine-rich peptide predominantly produced in theliver. This molecule regulates the absorption of iron in the intestineand inhibits release of iron from macrophages. Hepcidin was initiallyisolated as an amino acid (aa) peptide in human plasma and urineexhibiting antimicrobial activity (Krause et al. (2000) FEBS Lett 489,147-150; Park et al. (2001) J Biol Chem 276, 7806-7810). Hepcidin cDNAsencoding an 83 aa precursor in mice and an 84 aa precursor in rat andman, including a putative 24 aa signal peptide, were subsequentlyidentified searching for liver-specific genes that were regulated byiron (Pigeon et al. (2001) J Biol Chem 276, 7811-7819). A cDNA structurefor human hepcidin suggests that it is translated as an 84 amino acidprepropeptide that is amino terminally processed to a 60 amino acidresidue prohepcidin peptide, which is further processed into a 25 aminoacid hepcidin peptide. (Park et al. (2001).

Hepcidin expression is abolished in mice exhibiting iron-overload due tothe targeted disruption of upstream stimulatory factor 2 (Usf2) generesembling the same phenotype as found in hfe−/− mice (Nicolas G, et al(2001) Proc Natl Acad Sd USA 98, 8780-8785), leading to the conclusionthat this peptide plays a pivotal role in iron metabolism. In contrast,overexpression of hepcidin was shown to result in severe iron deficiencyanemia in transgenic mice (Nicolas et al. (2002) Proc Natl Acad Sci USA99, 4596-4601), indicating that hepcidin is a central regulator of ironhomeostasis. Moreover, recent studies have shown that liver hepcidinexpression is decreased in the hfe knockout mouse (Ahmad et al. (2002)Blood Cells Mci Dis 29, 361-366) and mutations in the hepcidin peptideare associated with severe juvenile hemochromatosis (Roetto et al.(2003) Nat Genet 33, 21-22), opening new perspectives in understandingof the molecular pathogenesis of iron overload. However, the mechanismby which hepcidin balances the body iron stores or adjusts the dietaryiron absorption in physiologic and pathologic conditions still remainsto be identified.

In this respect, the cellular localization of this peptide and itsregulation in various iron states are of major importance in the studyof hepcidin function. Although Northern blot analysis of human and mousehepcidin mRNA levels in various organs revealed that hepcidin ispredominantly expressed in liver (Krause et al. (2000) FEBS Lett 489,147-150; Park et al. (2001) J Biol Chem 276, 7806-7810; Nicolas et al.(2002) Proc Natl Acad Sci USA 99, 4596-4601), no data exists on thecellular localization of this peptide.

SUMMARY OF THE INVENTION

The present invention concerns hepcidin regulation of iron uptake bymammalian cells and the use of hepcidin and/or hepcidin specificantibodies in the diagnosis of diseases involving disturbances of ironmetabolism. The diagnostic detection kits of the present invention canbe particularly useful in screening the overall population of eitherhumans or animals and identifying those subjects who have thesediseases.

One aspect of the invention is a method for diagnosing a diseasecondition characterized by non-physiological levels of hepcidin,comprising obtaining a tissue or fluid sample from a subject; contactingthe sample with an antibody or fragment thereof that specifically bindsto a polypeptide from the mid-portion (amino acids 20 to 50) orC-terminus (amino acids 65 to 84) of hepcidin of SEQ ID NO: 2, andquantifying the hepcidin level using an assay based on binding of theantibody and the polypeptide; wherein the non-physiological level ofhepcidin is indicative of the disease condition. In one aspect of thepresent invention, sensitive diagnostic methods and kits wereestablished enabling the detection of prohepcidin in human plasma. Theinvention opens a broad range of therapeutic perspectives, where ahepcidin antibody and diagnostic methods and kits can be used for thedetermination of hepcidin as a parameter for the progress of thediseases mentioned above during and after therapy.

One embodiment of the invention concerns the generation and purificationof a hepcidin protein, including prohepcidin and fragments thereof.Another embodiment of the invention concerns hepcidin specificantibodies, or fragments or variants thereof that, in turn, can be usedin immunoassays to detect a hepcidin protein, including prohepcidin insuspected humans or animals.

In another aspect of the invention, the hepcidin diagnostic methods andkits can be used in genetic technological approaches, such as foroverexpressing or downregulating hepcidin.

In still another aspect of the invention, hepcidin can be used intherapeutic treatment of the diseases described herein, by treatingsubjects with hepcidin, and agonists or antagonists of hepcidin. Ironuptake in cells could be modulated by varying the concentration ofhepcidin, inhibiting hepcidin binding to iron or to the TfR2 receptor.Accordingly, hepcidin, and agonists or antagonists of hepcidin may beuseful in the treatment of conditions where there is a disturbance iniron metabolism. For example, such substances may be useful in thetreatment of such aforementioned diseases.

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino-acid sequence of the human hepcidinprecursor protein containing a typical 24 aa signal peptide at theN-terminus (the line between aa 24 and 25 indicates the putative signalsequence cleavage site), a 35 aa pro-region, and the C-terminal 20-,22-, and 25-aa hepcidin peptides differing only by their N-terminaltruncation as denoted by arrows. After cleavage of the signal peptidefrom hepcidin precursor, the prohepcidin molecule is produced consistingof 60 aa. The proposed disulfide connectivity in hepcidin 25 is 1-8,2-7, 3-6, and 4-5 as shown by dashed lines (from Hunter et al., 20). Theantisera EG (1 and 2)-Hep N are raised against hepcidin precursor aa28-47 (SEQ ID NO: 3), antiserum EG(1)-HepC is raised against aa 70-84(SEQ ID NO: 4) as denoted by the antibody symbols.

FIG. 2 illustrates as follows: (A): RT-PCR analysis of human liver(lanes 2 and 3) and HepG2 cells (lanes 4 and 5) showing gene expressionof hepcidin. A by DNA ladder is indicated (lanes 1 and 7). Lane 6 showsa negative control. (B-D): Western blot analyses of hepcidin in extractsof guinea pig (lanes 1) and human liver lanes 2) as well as in HepG2cells (lanes 3), human serum (lanes 4), and guinea pig skeletal muscle(lanes 5, control) with antibodies EG(1)-HepN (B), EG (2)-HepN (C) andEG (1)-HepC (D). Note the immunoreactive bands at 10 and 20 kDa obtainedwith all antibodies recognizing different epitopes in the hepcidinprecursor. (Molecular mass markers used: phosphorylase B, 105 kDa;glutamic dehydrogenase, 53 kDa; carbonic anhydrase, 34 kDa;myoglobin-blue, 23 kDa; myoglobin-red, 17 kDa; lysozyme, 13 kDa;aprotinin, 7 kDa; insulin, 3 kDa.)

FIG. 3 illustrates detection of hepcidin in HepG2 cells byimmunofluorescence microscopy using the antibodies EG(1)-HepN (A),EG(2)-HepN (B), and EG(1)-HepC (C) (Scale bar 8 μm)

FIG. 4 illustrates the cellular localization of hepcidin in guinea pig(A-F) and human (G-I) liver. The paraffin sections immunostained withthe region-specific antibodies EG(1)-HepN (A, D, 25 G), EG(2)-HepN (B,E, H) and EG(1)-HepC (C, F, I) show a distinct immunoreactivity at thebasolateral membrane domain of hepatocytes (arrows). (Magnification:A-C, ×180; D-I, ×540).

FIG. 5 illustrates immunohistochemical sections of guinea pig liver (A,antibody EG(1)-HepN; B, antibody EG(2)-HepN: C, antibody EG(1)-HepCshowing the clear zonation of hepcidin within the hepatic lobules withdecreasing immunoreactivity from periportal zones (stars) towards thecentral veins (arrowheads). Note that no immunoreactivity is found inhepatocytes around the central veins. (The arrow in B indicates a portaltriad.) (A-C, ×180).

FIG. 6 illustrates ELISA results for circulating human prohepcidin. Arepresentative standard curve with concentrations of hepcidin-(28-47)(SEQ ID NO: 3) in ng/ml and the extinction of the ELISA solution at 450nm wavelength are shown. Note the high resolving power in the range of 4to 400 ng/ml hepcidin-(28-47).

FIG. 7 illustrates the complete nucleotide (SEQ ID NO: 1) and amino acidsequences (SEQ ID NO: 2) of one form of hepcidin reproduced from GenBankdatabase accession nos. NN021175 and AAH20612, respectively.

FIG. 8 illustrates as follows: (A) RT-PCR analysis of human (lane 2),mouse (lane 3), and rat (lane 4) kidney showing gene expression ofhepcidin. A by DNA ladder is indicated (lanes 1 and 5). (B, C) Westernblot analyses of hepcidin in extracts of human lanes 1), rat (lanes 2),and mouse (lanes 3) kidney, as well as in human urine (lanes 4) withantibodies EG(2)-HepN (B), and EG(1)-HepC (C). Note the immunoreactivebands at 9.5 kDa obtained with both antibodies recognizing differentepitopes in the hepcidin precursor. (Molecular mass markers used:phosphorylase B, 105 kDa; glutamic dehydrogenase, 53 kDa; carbonicanhydrase, 34 kDa; myoglobin-blue, 23 kDa; myoglobin-red, 17 kDa;lysozyme, 13 kDa; aprotinin, 7 kDa; insulin, 3 kDa)

FIG. 9 illustrates the cellular localization of hepcidin in rat renalcortex. The paraffin sections immunostained with the region-specificantibodies EG(1)-HepC (A), EG(2)-HepC (B), EG(1)-HepN (C), andEG(2)-HepN (D) show a distinct immunoreactivity in distal tubuli of therenal cortex. In some tubuli immunoreactivity is distributed within thecytoplasm of the epithelial cells (arrows), but in others theimmunoreactivity is localized at the apical pole of the respective cells(D, arrowheads). Note that glomeruli asterisks) lack any hepcidinimmunoreactivity. (Magnification: A, ×90; B-D, ×180)

FIG. 10 illustrates the tissue distribution of hepcidin in rat (A and C)and mouse (B and D) kidney. Immunohistochemistry with antibodyEG(2)-HepN shows the outer medulla (A and B) with marked decrease ofhepcidin immunoreactivity between the outer stripe (os) and inner stripe(is), which is indicated by black dotted arcs. C and D show the lack ofhepcidin immunoreactivity in the inner medulla (m). Strongimmunoreactivity is observed in the cortex (c). (Magnification: A, B,and D, ×90; C, ×180)

FIG. 11 illustrates the subcellular localization of hepcidin in the ratkidney with antibodies EG(1)-HepN (A), EG(2)-HepN (B and D), andEG(1)-HepC (C). In some distal tubuli hepcidin immunoreactivity isdistributed within the cytoplasm of the epithelial cells (arrows), butin others the immunoreactivity is strongly concentrated toward theapical pole of the respective cells (black arrowheads). Note thatglomeruli (asterisk) and proximal tubuli (transparent arrowheads) lackany hepcidin immunoreactivity. (Magnification: A-D, ×360)

FIG. 12 illustrates the cellular localization of hepcidin in humankidney. Antibodies EG(1)-HepN (A), EG(2)-HepN (B and D), and EG(1)-HepC(C) show distinct immunoreactivity in distal tubuli of the renal cortex(arrows). In the same tubuli, intercellular differences of hepcidinimmunoreactivity exist showing strongly (black arrowheads) and faintlyimmunoreactive (transparent arrowheads) epithelial cells withcytoplasmic staining. No immunoreactivity is seen in the glomeruli(asterisks). (Magnification: A-C, ×180; D, ×360)

FIG. 13 illustrates the detection of hepcidin immunoreactivity at theapical pole of distal tubuli cells in human kidney with antibodiesEG(1)-HepN (A), EG(2)-HepN (B and D), and EG(2)-Hep C(C). Note thestrong immunostaining at the apical pole of the secretory epithelialcells (black arrowheads, some cells lack hepcidin immunoreactivity(transparent arrowheads). The asterisk indicate a glomerulus.(Magnification: A-C, ×180; 0, ×360)

FIG. 14 illustrates box-plot of values of venous serum and urinepro-hepcidin concentrations in 22 healthy volunteers (control) and 22patients with chronic renal insufficiency. The line within the boxindicates the median, and the circle indicates the mean. The lower andupper edge of the box indicates the 1st and 3rd quartile, the whiskersthe minimum and maximum values. The dashed line marks the mean level ofthe control group for circulating immunoreactive pro-hepcidin (104.2ng/ml).

BEST MODE OF CARRYING OUT THE INVENTION

The present invention describes that hepcidin regulates iron uptake bymammalian cells and nonphysiological expression of hepcidin results indisease involved in distribution of iron metabolism. The term hepcidin,as used herein, means prohepcidin, hepcidin or fragments thereof. Thephysiological concentration of hepcidin in the blood is in the range ofabout 50 to about 150 ng/ml. Nonphysiological concentrations are belowor over this range. Nonphysiological amounts of hepcidin protein or afragment thereof are associated with disturbances of iron metabolism,resulting in iron deficiency Or overload, such as iron deficiencyanemia; genetic and nongenetic iron overload diseases, such ashemosiderosis and hemochromatosis or secondary hemochromatosis,aceruloplasminemia, hypotransferrinemia, atransferrinemia; iron overloaddiseases of undetermined origin, for instance in the case of diseases ofthe biliary system, liver diseases, especially alcoholic liver diseases,nonalcoholic steatohepatitis, and chronic hepatitis B and C infections;diseases of utilization of iron, such as sideroblastic anemia,thalassemia; hematologic diseases, such as leukemia, polyglobulie,macrocytic, microcytic or normocytic anemia, anemia withreticulocytosis, hemolytic anemia; disturbances of thereticuloendothelial system due to infections and diseases; inflammationsand infections, including sepsis; immunologic diseases and tumors, suchas carcinoma, sarcoma, lymphoma, that result in non-physiologic hepcidinconcentrations; neurodegenerative diseases, such as Alzheimer's diseaseand Wilson's disease. This discovery has permitted the development ofassays for a hepcidin protein and fragments thereof and their subsequentpurification with retention of their native configuration andphysiological activity. The invention is based, in part, on thediscovery that in patients suffering from certain disorders a hepcidinprotein is present in tissue, blood and body fluid of a human or animal.

This invention provides the first demonstration that a hepcidin protein,including prohepcidin in subjects of these disorders are present inhuman or animal tissue, blood and body fluids in concentrations greatlyexceeding that found in normal humans or animals that are not subjectsof these disorders. This is achieved by examining a sample of tissue,blood, or body fluid from a patient, and detecting the presence andquantity of hepcidin protein and/or prohepcidin. The detection andquantitative measurement of any hepcidin protein, including prohepcidinor fragment thereof in tissue, blood or body fluids in accordance withthis invention is useful in confirming a clinical diagnosis of thediseases described herein, in affected patients and in following thecourse of the disease. The invention is also useful in monitoring thedisease during and subsequent to a period of treatment with agents thatare being tested for their ability to stabilize, decrease or prevent theoccurrence of such diseases.

For purposes of description only, the invention will be described interms of: (a) generating a hepcidin protein, including prohepcidin orfragments thereof; (b) generating antibodies that specifically bind ahepcidin protein, including prohepcidin or fragments thereof; (c)diagnostic assays and kits for diagnosing subtyping or monitoring thediseases described herein; (d) methods for over expressing and downregulating hepcidin or prohepcidin; and (e) therapeutic treatment of thediseases described herein.

In one aspect of the invention, Applicants provide a method fordetermining the role of hepcidin in physiologic conditions and inrelevant diseases. In another aspect of the invention Applicants providespecific antibodies against the midportion and the C terminus of thehepcidin precursor molecule. In this aspect of the invention, theseantibodies were used to define the cellular localization of hepcidin inthe human and guinea pig liver. A sensitive ELISA was established, whichdetects prohepcidin in human serum of patients with HH, chronic renalinsufficiency (CRI) and renal anemia (RA). Applicants have describedthat prohepcidin is released across the hepatocyte basolateral membraneinto the blood and is subjected to renal elimination. Since the serumlevels of hepcidin are remarkably downregulated in HH and chronic RA,hepcidin must play a role in the pathophysiology of these diseases.

Production of a Hepcidin Protein Isolating a Hepcidin Protein from Bloodand Body Fluids

For purposes of the present invention the term hepcidin protein isdefined as any mammalian hepcidin polypeptide sharing about 80 percentamino acid sequence identity with the predicted amino acid sequencepublished by Pigeon and co-workers ((2001) J. Biol. Chem. 276,7811-7819). The hepcidin proteins provided herein include prohepcidin,hepcidin and fragments thereof. The hepcidin proteins provided hereinalso include proteins characterized by amino acid sequences similar tothose of purified hepcidin proteins but into which modification arenaturally provided or deliberately engineered. For example,modifications in a hepcidin peptide or DNA sequences can be made bythose skilled in the art using known techniques. Modifications ofinterest in a hepcidin protein sequence may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein. Regions of a hepcidinprotein that are important for the protein function can be determined byvarious methods known in the art including the alanine-scanning methodwhich involved systematic substitution of single or strings of aminoacids with-alanine, followed by testing the resulting alanine-containingvariant for biological activity. This type of analysis determines theimportance of the substituted amino acid(s) in biological activity.

Production of a hepcidin protein may be accomplished by isolating ahepcidin protein from the tissue, blood or body fluids of humans oranimals suffering from hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and other such diseases described herein,using standard techniques known by those of skill in the art. Suchtechniques included in the invention also relate to methods forproducing a hepcidin protein comprising growing a culture of host cellsin a suitable culture medium, and purifying a hepcidin protein from thecells or the culture in which the cells are grown.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated hepcidin proteins of the present invention. Forexample, a hepcidin protein can also be produced by chemical synthesisof the amino acid sequence of a hepcidin protein (Pigeon et al., (2001)J. Bid. Chem. 276, 7811-7819), as predicted from the cloning andsequencing of a cDNA coding for a hepcidin protein. This hepcidinprotein sequence information may be utilized to predict the appropriateamino sequence of a fragment of a hepcidin protein to be chemicallysynthesized using standard peptide synthesis methods known in the art.These methods include a solid-phase method devised by R. BruceMerrifield, (Erickson and Merrifield, “Solid-Phase Peptide Synthesis”,in The Proteins, Volume 2, H. Neurath & R. Hill (Eds.) Academic Press,Inc., New York pp. 255-257; Merrifield, (1986) “Solid phase synthesis”,Science, 242:341-347). In the solid-phase method, amino acids are addedstepwise to a growing peptide chain that is linked to an insolublematrix, such as polystyrene beads. A major advantage of this method isthat the desired product at each stage is bound to beads that can berapidly filtered and washed and thus the need to purify intermediates isobviated. All of the reactions are carried out in a single vessel, whicheliminates losses due to repeated transfers of products. This solidphase method of chemical peptide synthesis can readily be automatedmaking it feasible to routinely synthesize peptides containing about 50residues in good yield and purity (Stewart and Young, (1984) Solid PhasePeptide Synthesis, 2nd ed., Pierce Chemical Co.; Tam et al., (1983) J.Am. Chem. Soc., 105:6442). For example, a hepcidin protein fragmentcorresponding to amino acid residues 1 to 50, or 34 to 84 as depicted inFIG. 47 could be synthesized. At the simplest level, commerciallyavailable peptide synthesizers are particularly useful in producingsmall peptides and fragments of a hepcidin protein. Fragments areuseful, for example, in generating antibodies against the nativehepcidin protein.

One skilled in the art can readily follow known methods for isolatingproteins in order to obtain one of the isolated hepcidinproteins/peptides of the present invention. These include, but are notlimited to, immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography, and immuno-affinity chromatography. See,e.g., Scopes, Protein Purification: Principles and Practice,Springer-verlag (1994); Sambrook, et al., in Molecular Cloning: ALaboratory Manual; Ausubel et al., Current Protocols in MolecularBiology.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a hepcidin protein. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a substantially homogeneous isolated recombinanthepcidin protein. A hepcidin protein thus purified is substantially freeof other mammalian proteins and is defined in accordance with thepresent invention as an isolated protein.

The sequence of a hepcidin protein may be identified using the Edmandegradation method of protein sequencing. This method sequentiallyremoves one amino acid residue at a time from the amino terminal end ofa peptide for subsequent sequence identification by chromatographicprocedures. See for example, the techniques described in Konigsberg andSteinman, (1977) Strategy and Methods of Sequence Analysis, in Neurathand Hill (eds.), The Proteins (3rd ed.) Vol. 3, pp. 1-178, AcademicPress. In addition, sequence analysis of a hepcidin protein may beaccelerated by using an automated liquid phase amino acid sequenatorfollowing described techniques (Hewick et al., (1981) J. Biol. Chem.,256:7990-7997; Stein and Undefriend, (1984) Analy. Chem., 136:7-23),thereby allowing for the analysis of picomolar quantities of a hepcidinprotein.

The purified hepcidin protein can be used in in vitro binding assaysthat are well known in the art to identify molecules that bind to ahepcidin protein. These molecules include but are not limited to, fore.g., small molecules, molecules from combinatorial libraries,antibodies or other proteins. The molecules identified in the bindingassay are then tested for agonist or antagonist activity in in vivotissue culture or animal models that are well known in the art. Inbrief, the molecules are titrated into a plurality of cell cultures oranimals and then tested for either cell/animal death or prolongedsurvival of the animal/cells.

In addition, the binding molecules may be complexed with toxins, e.g.,ricin or cholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to a tumor or other cellby the specificity of the binding molecule for a hepcidin protein.

Cloning and Expression of Recombinant Hepcidin Protein

In other embodiments, production of a hepcidin protein can be achievedby recombinant DNA technology. For example, appropriate hepcidinnucleotide coding sequences may be synthesized, cloned and expressed inappropriate host cells. Since the DNA sequence coding for a hepcidinprotein is known (Pigeon et al., (2001) J. Bid. Chem. 276, 7811-7819),DNA probes may be synthesized by standard methods known in the art toscreen cDNA libraries prepared from liver tissue from human or animalsubjects suffering from hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and other diseases described herein, forspecific hepcidin protein cDNA's. These DNA probes can further be usedto isolate the entire family of hepcidin protein genes from these cDNAlibraries using methods that are well known to those skilled in the art.See, for example, the techniques described in Maniatis et al., (1982)Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y., Chapter 7.

Hybridization procedures are useful for the screening of recombinantclones by using labeled mixed synthetic oligonucleotide probes whereeach probe is potentially the complete complement of a specific DNAsequence in the hybridization sample that includes a heterogeneousmixture of denatured double-stranded DNA. For such screening,hybridization is preferably performed on either single-stranded DNA ordenatured double-stranded DNA. By using stringent hybridizationconditions directed to avoid non-specific binding, it is possible, forexample, to allow the autoradiographic visualization of a specific DNAclone by the hybridization of the target DNA to that single probe in themixture which is its complete complement (Wallace, et al., (1981)Nucleic Acids Research, 9:879)

Alternatively, an expression library can be screened indirectly for ahepcidin protein of the invention having at least one epitope usingantibodies to the protein. Such antibodies can both be polyclonally ormonoclonally derived and used to detect an expression product indicativeof the presence of a hepcidin protein. Generally, a lambda gtl1 libraryis constructed and screened immunologically according to the method ofHuynh, et al., 1985) (in DNA Cloning: A Practical Approach, D. M.Glover, ed., 1:49)

The development of specific DNA sequences encoding a hepcidin proteincan also be obtained by: (1) isolation of a double stranded DNA sequencefrom the genomic DNA, and (2) chemical manufacture of a DNA sequence toprovide the necessary codons for the protein of interest.

The polymerase chain reaction (PCR) technique can be utilized to amplifythe individual members of a hepcidin family for subsequent cloning andexpression of hepcidin protein cDNA5 (e.g., see U.S. Pat. Nos.4,683,202; 4,683,195; 4,889,818; Gyllensten et al., (1988) Proc. Nat'lAcad. Sci. USA, 85:7652-7656; Ochman et al., (1988) Genetics,120:621-623; Triglia et al., (1988) Nuci. Acids. Res., 16:8156; Frohmanet al., (1988) Proc. Nat'l Acad. Sci. USA, 85:8998-9002; Loh et al.,(1989) Science, 243:217-220).

Methods that are well known to those skilled in the art can be used toconstruct expression vectors containing a hepcidin protein or fragmentsthereof coding sequences and appropriate transcriptional/translationalcontrol signals. These methods include in vitro recombinant DNAtechniques, synthetic techniques and in vivo recornbination/geneticrecombination. See, for example, the techniques described in Maniatis etal., 1982, Molecular Cloning A Laboratory Manual, Cold Spring HarborLaboratory, N.Y., Chapter 12.

A variety of host-expression vector systems may be utilized to express ahepcidin protein or fragment thereof. These include but are not limitedto microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a coding sequence for a hepcidin protein or fragmentsthereof; yeast transformed with recombinant yeast expression vectorscontaining a coding sequence for a hepcidin protein or fragment thereof;insect cell systems infected with recombinant virus expression vectors(e.g., baculovirus) containing a coding sequence for a hepcidin proteinor fragment thereof; or animal cell systems infected with recombinantvirus expression vectors (e.g., adenovirus, vaccinia virus) containing acoding sequence for a hepcidin protein or fragment thereof.

The expression elements of these vectors vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage lambda, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedrin promoter may beused; when cloning in mammalian cell systems, promoters such as theadenovirus late promoter or the vaccinia virus 7.5K promoter may beused. Promoters produced by recombinant DNA or synthetic techniques mayalso be used to provide for transcription of the inserted codingsequence for a hepcidin protein or fragment thereof.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For reviews see, Current Protocols in MolecularBiology, Vol. 2, (1988) Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience Ch. 13; Grant et al., (1987) Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, (1987)Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, (1986) DNA Cloning,Vol. II, IRL Press, Wash., D.C. Ch. 3; and Bitter, (1987) HeterologousGene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y., Vol. 152, Pp. 673-684; and The Molecular Biology ofthe Yeast Saccharomyces, (1982) Eds. Strathern et al., Cold SpringHarbor Press, Vols. I and II. For complementation assays in yeast, cDNA5for hepcidin proteins or fragments thereof may be cloned into yeastepisomal plasmids (YEp) that replicate autonomously in yeast due to thepresence of the yeast 2 mu circle. A hepcidin protein or fragmentthereof sequence may be cloned behind either a constitutive yeastpromoter such as ADH or LEU2 or an inducible promoter such as GAL(Cloning in Yeast, Ch. 3, R. Rothstein (1986) In DNA Cloning Vol. 11, APractical Approach, Ed. DM Glover, IRL Press, Wash., D.C.). Constructsmay contain the 5′ and 3′ non-translated regions of a cognate hepcidinprotein mRNA or those corresponding to a yeast gene. YEp plasmidstransform at high efficiency and the plasmids are extremely stable.Alternatively vectors may be used which promote integration of foreignDNA sequences into the yeast chromosome.

A particularly good expression system that could be used to express ahepcidin protein or fragments thereof is an insect system. In one suchsystem, Autographa californica nuclear polyhedrosis virus (AcNPV) isused as a vector to express foreign genes. The virus grows in Spodopterafrugiperda cells. A hepcidin protein or fragment thereof coding sequencemay be cloned into nonessential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of the polyhedringene results in production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (e.g., see Smith et al.,(1983) J. Biol., 46:586; Smith, U.S. Pat. No. 4,215,051). In addition,materials and methods for baculovirus/insect cell expression systems arecommercially available in kit form from, e.g., Invitrogen, San Diego,Calif., U.S.A. (the MaxBat' kit), and such methods are well known in theart, as described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987), incorporated herein by reference. Asused herein, an insect cell capable of expressing a hepcidinpolynucleotide of the present invention is transformed.

In cases where an adenovirus is used as an expression vector, a hepcidinprotein or fragment thereof coding sequence may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vivo or in vitro recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing a hepcidin protein of fragment thereof in infected hosts.(e.g., See Logan & Shenk, (1984) Proc. Natl. Acad. Sci., (USA)81:3655-3659). Alternatively, the vaccinia 7.5K promoter may be used.(e.g., see Mackett et al., (1982) Proc. Natl. Acad. Sci., (USA)79:7415-7419; Mackett et al., (1984) J. Virol., 49:857-864; Panicali etal., (1982) Proc. Natl. Acad. Sci., 79: 4927-4931).

Specific initiation signals may also be required for efficienttranslation of the inserted hepcidin protein or fragment thereof codingsequences. These signals include the ATG initiation codon and adjacentsequences. In cases where the entire hepcidin protein genome, includingits own initiation codon and adjacent sequences, are inserted into theappropriate expression vectors, no additional translational controlsignals may be needed. However, in cases where only a portion of ahepcidin protein coding sequence is inserted, exogenous translationalcontrol signals, including the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of a hepcidin protein or fragment thereof coding sequence toensure translation of the entire insert. These exogenous translationalcontrol signals and initiation codons can be of a variety of origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bitter et al., (1987) Methods inEnzymol., 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression driven by certainpromoters can be elevated in the presence of certain inducers, (e.g.,zinc and cadmium ions for metallothionein promoters). Therefore,expression of the genetically engineered hepcidin protein or fragmentthereof may be controlled. This is important if the protein product ofthe cloned foreign gene is lethal to host cells. Furthermore,modifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed.

The host cells which contain a hepcidin protein or fragment thereofcoding sequence and which express the biologically active hepcidinprotein or fragment thereof gene product may be identified by at leastfour general approaches: (a) DNA-DNA hybridization; (b) the presence orabsence of “marker” gene functions; (c) assessing the level oftranscription as measured by expression of hepcidin protein mRNAtranscripts in host cells; and (d) detection of hepcidin protein geneproducts as measured by immunoassays or by its biological activity.

In the first approach, the presence of a hepcidin protein or fragmentthereof coding sequence inserted in the expression vector can bedetected by DNA-DNA hybridization using probes comprising nucleotidesequences that are homologous to a hepcidin protein coding sequence orparticular portions thereof substantially as described recently (Pigeonet al., (2001) J. Biol. Chem. 276, 7811-7819)

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if a hepcidin protein or fragment thereof coding sequence is insertedwithin a marker gene sequence of the vector, recombinants containing ahepcidin protein or fragment thereof coding sequence can be identifiedby the absence of the marker gene function. Alternatively, a marker genecan be placed in tandem with a hepcidin protein or fragment thereofcoding sequence under the control of the same or different promoter usedto control the expression of a hepcidin coding sequence. Expression ofthe marker in response to induction or selection indicates expression ofa hepcidin protein coding sequence.

In the third approach, transcriptional activity for a hepcidin proteinor fragment thereof coding region can be assessed by hybridizationassays. For example, RNA can be isolated and analyzed by Northern blotusing a probe homologous to a hepcidin protein or fragment thereofcoding sequence or particular portions thereof substantially asdescribed (Pigeon et al., (2001) J. Biol. Chem. 276, 7811-7819).Alternatively, total nucleic acids of the host cell may be extracted andassayed for hybridization to such probes.

In the fourth approach, the expression of a hepcidin protein or fragmentthereof product can be assessed immunologically, for example by Westernblots, immunoassays such as radioimmunoprecipitation, enzyme-linkedimmunoassays and the like.

Once a recombinant that expresses a hepcidin protein or fragment thereofis identified, the gene product should be analyzed. This can be achievedby assays based on the physical, immunological or functional propertiesof the product. For example, the methods of the invention include aprocess for producing a hepcidin protein in which a host cell containinga suitable expression vector that includes a hepcidin polynucleotide ofthe invention is cultured under conditions that allow expression of theencoded hepcidin protein. A hepcidin protein can be recovered from theculture, conveniently from the culture medium, or from a lysate preparedfrom the host cells and further purified. Preferred embodiments includethose in which the protein produced by such process is a full length ormature form of the protein.

The present invention further provides isolated hepcidin protein encodedby the nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” is intended nucleotide fragments that differ from anucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical protein sequence. Preferred nucleic acid fragmentsof the present invention are the Orbs that encode proteins.

A hepcidin protein of the present invention can alternatively bepurified from cells that have been altered to express a hepcidinprotein. As used herein, a cell is altered to express a desiredpolypeptide or protein when the cell, through genetic manipulation, ismade to produce a hepcidin protein which it normally does not produce orwhich the cell normally produces at a lower level. One skilled in theart can readily adapt procedures for introducing and expressing eitherrecombinant or synthetic sequences into eukaryotic or prokaryotic cellsin order to generate a cell which produces a hepcidin protein of thepresent invention.

A hepcidin protein of the invention may also be expressed as a productof transgenic animals, e.g., as a component of the milk of transgeniccows, goats, pigs, or sheep which are characterized by somatic or germcells containing a nucleotide sequence encoding a hepcidin protein.

A hepcidin protein may also be produced by known conventional chemicalsynthesis. Methods for constructing a hepcidin protein of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed hepcidin protein sequences, by virtue ofsharing primary, secondary or tertiary structural and/or conformationalcharacteristics with natural hepcidin protein may possess biologicalproperties in common therewith, including protein activity. Thus, theymay be employed as biologically active or immunological substitutes fora natural, purified hepcidin protein in screening of therapeuticcompounds and in immunological processes for the development ofantibodies.

A hepcidin protein of the invention may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant protein. The resulting expressed hepcidin protein may thenbe purified from such culture (i.e., from culture medium or cellextracts) using known purification processes, such as gel filtration andion exchange chromatography. The purification of a hepcidin protein mayalso include an affinity column containing agents which will bind to theprotein; one or more column steps over such affinity resins asconcanavalin Aagarose, heparin-toyopearl or Cibacrom blue 3GA Sepharoseone or more steps involving hydrophobic interaction chromatography usingsuch resins as phenyl ether, butyl ether, or propyl ether; orimmunoaffinity chromatography.

Alternatively, a hepcidin protein of the invention may also be expressedin a form that will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a Histag. Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. A hepcidinprotein can also be tagged with an epitope and subsequently purified byusing a specific antibody directed to such epitope. One such epitope(“FLAG®”) is commercially available from Kodak (New Haven, Conn.).

Other fragments and derivatives of the sequences of hepcidinproteins/peptides which would be expected to retain protein activity inwhole or in part (e.g., binding to a TfR2 receptor, binding to ahepcidin specific antibody, etc.) and are useful for screening or otherimmunological methodologies may also be easily made by those skilled inthe art given the disclosures herein. Such modifications are encompassedby the present invention.

A hepcidin protein or fragment thereof should be immunoreactive whetherit results from the expression of the entire gene sequence, a portion ofthe gene sequence or from two or more gene sequences which are ligatedto direct the production of chimeric proteins. This reactivity may bedemonstrated by standard immunological techniques, such asradioimmunoprecipitation, radioimmune competition, or immunoblots.

Generation of Antibodies which Define a Hepcidin Protein or FragmentThereof

Various procedures known in the art may be used for the production ofantibodies to the mid-portion (amino acids 20 to 50) or C-terminus ofepitopes (amino acids 65 to 84) of a hepcidin protein of SEQ ID NO: 2.The hepcidin specific antibodies bind those epitopes and no other knownsequences. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments and an Fab expressionlibrary. For the production of antibodies, various host animals may beimmunized by injection with a particular hepcidin protein, or asynthetic hepcidin protein, including but not limited to rabbits, mice,rats, etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum.

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbits, mice, or rats. Briefly, hepcidin is utilized toimmunize the animal through intraperitoneal, intramuscular, intraocular,or subcutaneous injections, an adjuvant such as Freund's complete orincomplete adjuvant. Following several booster immunizations, samples ofserum are collected and tested for reactivity to hepcidin. Particularlypreferred polyclonal antisera will give a signal on one of these assaysthat is at least three times greater than background. Once the titer ofthe animal has reached a plateau in terms of its reactivity to hepcidin,larger quantities of antisera may be readily obtained either by weeklybleedings, or by exsanguinating the animal.

Monoclonal antibodies to peptides of hepcidin may be prepared by usingany technique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Kohier and Milstein,(Nature, (1975) 256:495-497), the more recent human B-cell hybridomatechnique (Kosbor et al., (1983) Immunology Today, 4:72) and theEBV-hybridoma technique (Cole et al., (1985) Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additionalembodiment of the invention monoclonal antibodies specific to hepcidinproteins/peptides may be produced in germ-free animals utilizing recenttechnology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridotnas(Cote at al., (1983) Proc. Natl. Acad. Sci., 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., (1985)in, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., (1984) Proc. Natl.Acad. Sci., 8 1:6851-6855; Neuberger et al., (1984) Nature, 312:604-608;Takeda et al., (1985) Nature, 314:452-454) by splicing the genes from amouse antibody molecule of appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biological activitycan be used; such antibodies are due to this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce hepcidin protein-specific single chain antibodies.

An additional embodiment of the invention utilizes the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,(1989) Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity to hepcidinproteins/peptides.

Antibody fragments that contain specific binding sites for a hepcidinprotein may be generated by known techniques. For example, suchfragments include but are not limited to: the F(ab′)₂ fragments whichcan be produced by pepsin digestion of the antibody molecule and the Fabfragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments.

Diagnostic Assays and Kits

Yet another purpose of the present invention is to provide reagents foruse in diagnostic assays for the detection of a hepcidin protein fromindividuals suffering from hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and such other diseases described herein.

In one mode of this embodiment, a hepcidin protein of the presentinvention may be used as an antigen in immunoassays for the detection ofthose individuals suffering from hemochromotosis, iron deficiencyanemia, hemosiderosis, liver cirrhosis and such other diseases describedherein. A hepcidin protein, polypeptide and/or peptide of the presentinvention may be used in any immunoassay system known in the artincluding, but not limited to: radioimmunoassays, enzyme-linkedimmunosorbent assay, “sandwich” assays, precipitin reactions, geldiffusion immunodiffusion assays, agglutination assays, fluorescentimmunoassays, protein A immunoassays and immunoelectrophoresis assays,to name but a few. U.S. Pat. No. 4,629,783 and patents cited thereinalso describe suitable assays.

According to the present invention, monoclonal or polyclonal antibodiesproduced to various forms of a hepcidin protein, can be used in animmunoassay on samples of blood, spinal fluid or other body fluid todiagnose subjects with hemochromotosis, iron deficiency anemia,hemosiderosis, liver cirrhosis and other diseases described herein.

In one embodiment of the invention, a sample of blood is removed fromthe patient by venesection and placed in contact with an anticoagulantsuch as EDTA, mixed, centrifuged at 600 g for 10 min and the plasmaremoved as is common in the art or a sample of spinal fluid is removedfrom the patient by lumbar puncture.

The antibodies described herein may be used as the basic reagents in anumber of different immunoassays to determine the presence of a hepcidinprotein in a sample of tissue, blood or body fluid. Generally speaking,the antibodies can be employed in any type of immunoassay, whetherqualitative or quantitative. This includes both the two-site sandwichassay and the single site immunoassay of the non-competitive type, aswell as in traditional competitive binding assays.

Particularly preferred, for ease of detection, and its quantitativenature, is the sandwich or double antibody assay, of which a number ofvariations exist, all of which are intended to be encompassed by thepresent invention.

For example, in a typical forward sandwich assay, unlabeled antibody isimmobilized on a solid substrate, e.g., microtiter plate wells, and thesample to be tested is brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen binary complex, a secondantibody, labelled with a reporter molecule capable of inducing adetectable signal, is then added and incubation is continued allowingsufficient time for binding with the antigen at a different site and theformation of a ternary complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal, which may be quantitated bycomparison with a control sample containing known amounts of antigen.Variations on the forward sandwich assay include the simultaneous assay,in which both sample and antibody are added simultaneously to the boundantibody, or a reverse sandwich assay in which the labelled antibody andsample to be tested are first combined, incubated and added to theunlabelled surface bound antibody. These techniques are well known tothose skilled in the art, and the possibility of minor variations willbe readily apparent. As used herein, “sandwich assay” is intended toencompass all variations on the basic two-site technique.

For the sandwich assays of the present invention, the only limitingfactor is that both antibodies have different binding specificities fora hepcidin protein. Thus, a number of possible combinations arepossible.

As a more specific example, in a typical forward sandwich assay, aprimary antibody is either covalently or passively bound to a solidsupport. The solid surface is usually glass or a polymer, the mostcommonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinylchloride or polypropylene. The solid supports maybe in the form of tubes, beads, discs or microplates, or any othersurfaces suitable for conducting an immunoassay. The binding processesare well known in the art. Following binding, the solid phase-antibodycomplex is washed in preparation for the test sample. An aliquot of thebody fluid containing a hepcidin protein to be tested is then added tothe solid phase complex and incubated at 25° C. for a period of timesufficient to allow binding of any hepcidin protein present to theantibody specific for hepcidin protein. The second antibody is thenadded to the solid phase complex and incubated at 25° C. for anadditional period of time sufficient to allow the second antibody tobind to the primary antibody-antigen solid phase complex. The secondantibody is linked to a reporter molecule, the visible signal of whichis used to indicate the binding of the second antibody to any antigen inthe sample. By “reporter molecule”, as used in the present specificationis meant a molecule which by its chemical nature, provides ananalytically detectable signal which allows the detection ofantigen-bound antibody. Detection must be at least relativelyquantifiable, to allow determination of the amount of antigen in thesample, this may be calculated in absolute terms, or may be done incomparison with a standard (or series of standards) containing a knownnormal level of antigen.

The most commonly used reporter molecules in this type of assay areeither enzymes or fluorophores. In the case of an enzyme immunoassay anenzyme is conjugated to the second antibody, often by means ofglutaraldehyde or periodate. As will be readily recognized, however, awide variety of different conjugation techniques exist, which are wellknown to the skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, β-galactosidase and alkaline phosphatase,among others. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidineare commonly used. It is also possible to employ fluorogenic substrates,which yield a fluorescent product rather than the chromogenic substratesnoted above. In all cases, the enzyme-labelled antibody is added to thefirst antibody-hepcidin protein complex and allowed to bind to thecomplex, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the tertiarycomplex of antibody-antigen-labeled antibody. The substrate reacts withthe enzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an evaluation of the amount of antigenthat is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein or rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.As in the enzyme immunoassay (EIA), the fluorescent-labelled antibody isallowed to bind to the first antibody-hepcidin protein complex. Afterwashing the unbound reagent, the remaining ternary complex is thenexposed to light of the appropriate wavelength, and the fluorescenceobserved indicates the presence of the antigen. Immunofluorescence andEIA techniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotopes, chemiluminescent or bioluminescentmolecules may also be employed. It will be readily apparent to theskilled artisan how to vary the procedure to suit the required use.

Alternatively, the sample to be tested either human blood or spinalfluid containing a hepcidin protein may be used in a single siteimmunoassay wherein it is adhered to a solid substrate either covalentlyor noncovalently. An unlabeled anti-hepcidin protein antibody is broughtinto contact with the sample bound on the solid substrate. After asuitable period of incubation, for a period of time sufficient to allowformation of an antibody-antigen binary complex a second antibody,labelled with a reporter molecule capable of inducing a detectablesignal, is then added and incubation is continued allowing sufficienttime for the formation of a ternary complex of antigen-antibody-labeledantibody. For the single site immunassay, the second antibody may be ageneral antibody (i.e., zenogeneic antibody to immunoglobulin,particularly anti-(IgM and IgG) linked to a reporter molecule) that iscapable of binding an antibody that is specific for a hepcidin proteinof interest.

A hepcidin gene (mutated or normal) can be utilized in an assay of ironmetabolism. The gene is expressed, with or without any accompanyingmolecules, in cell lines or primary cells derived from human or animalsubjects, healthy subjects, or cells from other organisms (such asrodents, insects, bacteria, amphibians, etc.). Uptake of iron by thesecells is measured, for example through the use of radioactive isotopes.Further, binding of iron to a hepcidin gene product can also bemeasured. Such experiments assist in assessing the role of a hepcidingene and hepcidin gene product in iron uptake, binding, and transport byand in cells.

Therapeutic Treatment

In one aspect of the invention, the hepcidin diagnostic methods and kitscan be used in genetic technological approaches, such as for overexpressing or down regulating hepcidin. In certain therapeuticapplications, it is desirable to down regulate the expression and/orfunction of a hepcidin gene, a mutant hepcidin gene, a hepcidin protein,or a mutant hepcidin protein. For example, down regulation of a normalhepcidin gene or a normal hepcidin protein is desirable in situationswhere iron is under accumulated in the body, for example in certainanemias (i.e., thalassaemias, hemolytic anemias, transfusions). On theother hand, down regulation of a mutant hepcidin gene or a hepcidinprotein is desirable in situations where iron is over accumulated in thebody.

As discussed above antibodies specific to a normal or a mutant hepcidinprotein can be prepared. Such antibodies can be used therapeutically inthe diseases described herein. For example, to block the action of amutant or normal hepcidin gene if the function associated with a mutantprotein is an up regulation of a normal hepcidin protein function andleads to an over accumulation of iron in the body. Similarly, antibodiescan be used therapeutically to block action of a hepcidin protein thatis causing an under accumulation of iron in the body.

In a similar manner, a hepcidin gene, either in a normal or in a mutantform, can be down regulated through the use of antisenseoligonucleotides directed against the gene or its transcripts. A similarstrategy can be utilized as discussed above in connection withantibodies. For a particularly valuable review of the designconsiderations and use of antisense oligonucleotides, see Uhlmann etal., (1990) Chemical Reviews 90:543-584, the disclosure of which ishereby incorporated by reference. The antisense oligonucleotides of thepresent invention may be synthesized by any of the known chemicaloligonucleotide synthesis methods. Such methods are generally described,for example, in Winnacker Chirurg (1992) 63:145. Antisenseoligonucleotides are most advantageously prepared by utilizing any ofthe commercially available, automated nucleic acid synthesizers. Onesuch device, the Applied Biosystems 380B DNA Synthesizer, utilizesbeta-cyanoethyl phosphoramidite chemistry.

Since the complete nucleotide synthesis of DNA complementary to ahepcidin gene is known, the mRNA transcript of the cDNA sequence is alsoknown. As such, antisense oligonucleotides hybridizable with any portionof such transcripts may be prepared by oligonucleotide synthesis methodsknown to those skilled in the art. While any length oligonucleotide maybe utilized in the practice of the invention, sequences shorter than 12bases may be less specific in hybridizing to the target mRNA, may bemore easily destroyed by enzymatic digestion, and may be destabilized byenzymatic digestion. Hence, oligonucleotides having 12 or morenucleotides are preferred. Long sequences, particularly sequences longerthan about 40 nucleotides, may be somewhat less effective in inhibitingtranslation because of decreased uptake by the target cell. Thus,oligomers of 12-40 nucleotides are preferred, more preferably 15-30nucleotides, most preferably 18-26 nucleotides. Sequences of 18-24nucleotides are most particularly preferred.

In still another aspect of the invention, hepcidin can be used in thetherapy of the disorders described herein, by treating subjects withhepcidin, and agonists or antagonists of hepcidin. Iron uptake in cellscan be modulated by varying the concentration of hepcidin, and/orinhibiting hepcidin binding to iron or to the transferrin receptor.Accordingly, hepcidin, and agonists or antagonists of hepcidin may beuseful in the treatment of conditions where there is a disturbance iniron metabolism. For example, such substances may be useful in thetreatment of conditions such as haemochromatosis, neurodegenerativediseases, ischemic tissue damage, including ischemic stroke or trauma,heart disease, and tumors, in particular skin cancer and such otherdiseases described herein.

The invention also contemplates methods of modulating iron metabolismusing hepcidin. In particular, the present invention relates to a methodfor treating conditions involving disturbances in iron metabolismcomprising administering an iron-modulating amount of hepcidin, or astimulant, agonist or antagonist of hepcidin. Conditions involvingdisturbances in iron metabolism which may be treated using the method ofthe invention include by way of example haemochromatosis,neurodegenerative diseases, ischemic tissue damage, including ischemicstroke or trauma, heart disease, and tumors, in particular skin cancerand such other diseases described herein. A substance which is anagonist or antagonist of hepcidin may be identified by determining theeffect of the substance on the binding activity of hepcidin and iron, orhepcidin and the transferrin receptors TfR1 or TfR2, or the effect ofthe substance on the expression of hepcidin in cells capable ofexpressing hepcidin including cells genetically engineered to expresshepcidin on their surface.

The invention therefore in one aspect relates to a method of identifyingagonists or antagonists of hepcidin comprising reacting a substancesuspected of being an agonist or antagonist of hepcidin with hepcidinand iron under conditions such that hepcidin is capable of binding toiron; measuring the amount of hepcidin bound to iron; and determiningthe effect of the substance by comparing the amount of hepcidin bound toiron with an amount determined for a control.

The invention also relates to a method of identifying agonists orantagonists of hepcidin comprising reacting a substance suspected ofbeing an agonist or antagonist of hepcidin with hepcidin and transferrinreceptor under conditions such that hepcidin is capable of binding tothe transferrin receptor; measuring the amount of hepcidin bound to atransferrin receptor; and determining the effect of the substance bycomparing the amount of hepcidin bound to a transferrin receptor with anamount determined for a control. The invention also relates to a methodof identifying agonists or antagonists of hepcidin comprising reacting asubstance suspected of being an agonist or antagonist of hepcidin with acell which produces hepcidin, measuring the amount of hepcidin expressedby the cell, and determining the effect of the substance by comparingthe amount of expression of hepcidin with an amount determined for acontrol. The invention further relates to a method for identifying anagonist or antagonist of hepcidinmediated iron uptake comprising:incubating a cell expressing hepcidin on its surface and a substancesuspected of being an agonist or antagonist of hepcidin in the presenceof iron and in the absence of transferrin, measuring the amount of ironuptake into the cell, and identifying an agonist or antagonist ofhepcidin-mediated iron uptake by comparing the amount of iron uptake inthe cell with the amount of iron uptake in a cell from a controlincubation in the absence of the substance.

In some embodiments of the invention, hepcidin peptides are provided fortherapeutic use in subjects having symptoms of a primary iron overloaddisease or syndrome, such as hemochromatosis, or other iron overloadcondition caused by secondary causes, such as repeated transfusions. Ahepcidin peptide can be full-length hepcidin or some fragment ofhepcidin. Preferably, a hepcidin peptide comprises the amino acidresidues 28 to 47 or 70 to 80 of a hepcidin (SEQ ID NO: 2). Thepredicted amino acid sequence and genomic and cDNA sequences of hepcidinwere provided in (Krause et al., (2000) FEBS Lett. 480, 147-150; Pigeonet al., (2001) J. Biol. Chem. 276, 7811-7819), hereby incorporated byreference in their entirety. A hepcidin protein or fragment thereof maybe administered with beta-2-microglobulin, such as in the form of acomplex. In some embodiments, a hepcidin protein greater than about 20amino acids is administered in a complex with beta-2-microglobulin.

In some embodiments of the invention, agonists or antagonists of ahepcidin protein or a transferrin receptor are provided. Agonists of ahepcidin polypeptide, and/or antagonists of a transferrin receptor, areuseful for example, in the treatment of primary or secondary ironoverload diseases or syndromes, while antagonists of a hepcidinpolypeptide, or agonists of the transferrin receptor are useful, forexample, in the treatment of iron deficiency conditions, such asanemias. In other embodiments, mutant hepcidin proteins/peptides areprovided which function as antagonists of the wild-type hepcidinprotein. Antagonists or agonists can also be antibodies, directedagainst a transferrin receptor, or the mid-portion (amino acids 20 to50) or C-terminal region (amino acids 65 to 84) of a hepcidin protein(SEQ ID NO: 2). In some embodiments of the invention, hepcidinpolypeptides can serve as antagonists of a transferrin receptor. Infurther embodiments of the invention, peptidomimetics can be designedusing techniques well known in the art as antagonists or agonists of ahepcidin protein and/or a transferrin receptor.

Ligands for a transferrin receptor, whether antagonists or agonists, canbe screened using the techniques described herein for the ability tobind to a transferrin receptor. Additionally, competition for hepcidinbinding to a transferrin receptor can be done using techniques wellknown in the art. Ligands, or more generally, binding partners for ahepcidin protein can be screened, for example, for the ability toinhibit the complexing of a hepcidin polypeptide tobeta-2-microglobulin, using techniques described herein.

In some embodiments of the invention, agonists or antagonists oftransferrin are similarly utilized to increase or decrease the amount ofiron transported into a cell, such as into a patient's hepatocytes orlymphocytes. For example, the efficacy of a drug, therapeutic agent,agonist, or antagonist can be identified in a screening program in whichmodulation is monitored in in vitro cell systems. Host cell systems thatexpress various mutant hepcidin proteins/peptides and are suited for useas primary screening systems. Candidate drugs can be evaluated byincubation with these cells and measuring cellular functions dependenton a hepcidin gene or by measuring proper hepcidin protein folding orprocessing. Such assays might also entail measuring receptor-likeactivity, iron transport and metabolism, gene transcription or otherupstream or downstream biological function as dictated by studies ofhepcidin gene function.

Alternatively, cell-free systems can be utilized. Purified hepcidinprotein can be reconstituted into artificial membranes or vesicles anddrugs screened in a cell-free system. Such systems are often moreconvenient and are inherently more amenable to high throughput types ofscreening and automation.

Criteria for the determination of the purity of a hepcidin proteininclude those standard to the field of protein chemistry. These includeN-terminal amino acid determination, one and two-dimensionalpolyacrylamide gel electrophoresis, and silver staining. The purifiedprotein is useful for use in studies related to the determination ofsecondary and tertiary structure, as aid in drug design, and for invitro study of the biological function of the molecule.

In some embodiments of the invention, drugs can be designed to modulatea hepcidin gene and a hepcidin protein activity from knowledge of thestructure and function correlations of a known hepcidin protein. Forthis, rational drug design by use of X-ray crystallography,computer-aided molecular modeling (CAMM), quantitative or qualitativestructure-activity relationship (QSAR), and similar technologies canfurther focus drug discovery efforts. Rational design allows predictionof protein or synthetic structures that can interact with and modify ahepcidin protein activity. Such structures may be synthesized chemicallyor expressed in biological systems. This approach has been reviewed inCapsey et al., Genetically Engineered Human Therapeutic Drugs, StocktonPress, New York (1988). Further, combinatorial libraries can bedesigned, synthesized and used in screening programs.

In order to administer therapeutic agents based on, or derived from, thepresent invention, it will be appreciated that suitable carriers,excipients, and other agents may be incorporated into the formulationsto provide improved transfer, delivery, tolerance, and the like.

A multitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, (15th Edition, Mack Publishing Company, Easton, Pa. (1975)),particularly Chapter 87, by Blaug, Seymour, therein. These formulationsinclude for example, powders, pastes, ointments, jelly, waxes, oils,lipids, anhydrous absorption bases, oil-in-water or water-in-oilemulsions, emulsions carbowax (polyethylene glycols of a variety ofmolecular weights), semisolid gels, and semi-solid mixtures containingcarbowax.

Any of the foregoing formulations may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive agent in the formulation is not inactivated by the formulationand the formulation is physiologically compatible.

The invention is not limited to the embodiments described herein and maybe modified or varied without departing from the scope of the invention.

EXAMPLES Example 1 Expression of Hepcidin in the Human Liver Tissues andTissue Preparation

Human liver samples (n=7) used in the present study were obtained afterhemi-hepatectomy in adult subjects with liver metastases. Healthytissues were fixed in 4% paraformaldehyde for immunohistochemistry orimmediately frozen in liquid nitrogen for RT PCR, Western blot andimmunofluorescence analysis.

Guinea pigs (n=7) and mice (n=S) were anesthetized and subsequentlysacrificed by cervical dislocation. Tissue specimens from liver,skeletal muscle and heart were resected and immediately frozen in liquidnitrogen for Western blot analysis or fixed in paraformaldehyde.

Peptide Synthesis, Immunization Procedure, and Antibodies.

From the published prohepcidin sequence (Krause et al., (2000) FEBSLett. 480, 147-150; Pigeon et al., (2001) J. Bid. Chem. 276, 7811-7819),the peptides hepcidin-(28-47) and hepcidin-(70-84) were synthesized as Cterminal amides using a standard Fmoc protocol (Cetin et al., (1994),Proc. Natl. Acad. Sci. USA 91, 2935-2939). Peptides were coupled tokeyhole limpet hemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimideester, and two SPF rabbits (Charles River If fa Credo) were immunizedwith each peptide conjugate (Eurogentec, Seraing, Belgium). Aftertesting the titer by ELISA, three antisera [EG(1)-HepC directed againsthepcidin(70-84) and EG(1)-HepN and EG(2)-HepN, each directed againsthepcidin-(28-47) were used in the present study (FIG. 1) (hepcidin28-47: PQQ TGQ LAE LQP QDR AGA RA SEQ. (SEQ ID NO: 3), hepcidin 70-84:CGC CHR SKC GMC CKT (SEQ ID NO: 4)). The peptide epitopes used for thegeneration of the antisera displayed no homology to any hithertoreported protein as confirmed by the BLAST P2 search.

The BT-TFR21 S antibody against mouse TfR2 (BioTrend, Cologne, Germany)was raised against the cytoplasmic N-terminus of mouse TfR2-alpha (TfR2)is alternatively spliced to alpha and beta isoforms, see Fleming et al.,(2000) Proc. Natl. Acad. Sci. USA 97, 2214-2219), showing 68% sequencehomology to the corresponding region of human TfR2-alpha. The antibodywas generated in rabbits and affinity purified.

Expression Analyses in the Human Liver.

RNA isolation was performed using Qiagen RNA easy kit including DNAdigestion. Reverse transcription (RT)-PCR analysis was performed asdescribed previously (Kulaksiz et al., (2002) Proc. Natl. Acad. Sci. USA99, 6796-6801; Kulaksiz et al., (2002) Am. J. Pathol. 161, 655-664)using the following primers and specifications given in 5-3′orientation: human hepcidin (GenBank database accession no. NM0211175),5′-CTG CAA CCC CAG GAC AGA G-3′ (SEQ ID NO: 5) and 5, GGA ATA AAT AAGGAA GGG AGG GG-3′, (SEQ ID NO: 6) corresponding to nucleotide positions147-165 and 338-316. Human TfR2 (#AFO6 7864), 5′-GAT TCA GGG TCA GGG AGGTG-3′ (SEQ ID NO: 7) and 5′-(GAA GGG GCT GTG ATT GAA GG-3′ (SEQ ID NO:8); corresponding to nucleotide positions 2496-2515 and 2694-2675. Afteran initial denaturation of 94° C. for 4 min; reactions were subjected to35 cycles of the following thermal program: 94° C. for 30 s, 60° C. for30 s, and 72° C. for 30 s; this program was followed by a final 5 minelongation step at 72° C. Amplification products were run on an ethidiumbromide-stained 1.8% 89 mM Tris/89 mM boric acid/2 mM EDTA (pH 8.3)agarose gel. The amplification of significant levels of genomic DNA wasexcluded by appropriate controls.

Expression Analyses in HepG2 Cells.

The human hepatoma HepG2 cells were obtained from the German Collectionof Microorganisms and Cell Culture (Braunschweig, Germany) and grown at37° C. in 5% CO2 in RPMI 1640 media (Gibco, Karisruhe, Germany)supplemented with 10% (vol/vol) heat-inactivated FBS, penicillin (100units/ml), and streptomycin (100 mg/ml). Cells were analyzed by RT PCRusing the primer specifications mentioned above. For immunofluorescencemicroscopy, HepG2 cells were grown on glass slides fixed for 4 min inmethanol, and pertneabilized with 0.5% Triton X-100 in PBS. Afterincubation with hepcidin (1:2000) and TfR2 antibodies (1:1000) for 60min, followed by incubation with Cy-3-conjugated anti-rabbit antibody(Dianova, Hamburg, Germany), the immunostaining was investigated underan Olympus AX70 microscope using appropriate filters.

Extraction of Hepcidin and TfR2 from Serum, Tissues and HepG2 Cells.

As a larger source of hepcidin Applicants used serum collected frompatients with chronic renal failure. For extraction of hepcidin, 20 mlserum samples were diluted 1:1 with 0.01 N HCl and adjusted to pH 3.0with concentrated HCl. Frozen tissues and HepG2 cells were mixed in 0.5M acetic acid and boiled for 8 min as described (Cetin et al. (1994)Proc Natl Acad Sci USA 91, 2935-2939; Cetin et al. (1995) Proc Natl AcadSd USA 92, 5925-5929) After homogenization with an Ultra-Turraxhomogenizer (Janke & Kunkei, Staufen, Germany) the samples werecentrifuged at 20,000×g for 20 min at 4° C. and the supernatants werefiltered through a 0.45-μm pore size filter. To enrich proteins, serumsamples, cell and total tissue extracts were applied to an octadecasilyl(C18) Sep-Pak cartridge (Waters, Mass.). The column was washed with 0.01M HCl and eluted with 30% (vol/vol) 2-propanol/30% (vol/vol)methanol/0.01 M HCl (Cetin et al. (1994) Proc Natl Acad Sci USA 91,2935-2939). Protein fractions were lyophilized and stored at 800 C untiluse. For TfR2 analysis, tissues and cells were homogenized in Tris-HClbuffer containing 100 mM NaCl, 50 mM Tris-HCl, pH 7.4, 10% glycerol, 1%Triton X-100, 2 mg/ml leupeptin, 2 mg/ml pepstatin, and 1 mMphenylmethylsulfonyl fluoride, and centrifuged at 100,000 g for 30 minat 4° C.

Immunoblot Analysis.

For Western blot analysis, protein extracts were incubated for 7 min at94° C. in sample buffer with 4% (wt/vol) SDS (Merck, Darmstadt,Germany), 50 mM Tris-HCl (pH 8.15), 1 mM EDTA, 3.24 mM dithiothreitol(Roth, Karisruhe, Germany), 12.5% (wt/vol) glycerol (Merck), and 0.002%bromophenol blue (Merck). To detect hepcidin, a 16.5%tricine-SDS-polyacrylamide gel was used according to the protocolspublished (Cetin et al., (1994), Proc. Natl. Acad. Sci. USA 91,2935-2939; Kulaksiz et al., (2002) Proc. Natl. Acad. Sd. USA 99,6796-6801; Kulaksiz et al., (2002) Am. J. Pathol. 161, 655-664; Cetin etal., (1995) Proc. Natl. Acad. Sci. USA 92, 5925-5929.) TfR2 immunoblotswere performed using 8% SDS-polyacrylamide gels. Followingelectrophoresis, proteins were transferred onto hydrophobicpolyvinylidene fluoride-based membranes (Pall, Portsmouth, England) bysemidry blotting. The membranes were incubated overnight with hepcidinor TfR2 antibodies at dilutions mentioned above. After washing inTris-buffered saline containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl,and 0.05% Tween 20, the respective immunoreactive proteins werevisualized after incubation with alkaline phosphatase-conjugated goatanti-rabbit antibody (diluted 1:50,000; Sigma) using nirro bluetetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as chromogens(Sigma). The immunoreaction on the Western blot was specifically blockedafter preincubation of the antibodies with the corresponding peptideimmunogens. Crossreactivity with the second goat anti-rabbit antibodywas excluded by appropriate controls (Cetin et al. (1994) Proc Natl AcadSci USA 91, 2935-2939; Kulaksiz et al. (2002) Proc Natl Acad Sci USA 99,6796-6801; Kulaksiz et al. (2002) Am J Pathol 161, 655-664; Cetin et al.(1995) Proc Natl Acad Sci USA 92, 5925-5929)

Immunohistochemistry and Immunofluorescence

Tissues were fixed in 4% paraformaldehyde for 18 h at 4° C. Afterdehydration in graded ethanol series, the specimens were embedded inparaffin. Paraffin sections (5 μm) were immunostained for hepcidin(antibodies EG(1)-HepN, EG(2)-HepN, and EG(1)-HepC, each diluted 1:2000)or TfR2 (antibody BT-TFR21-S. diluted 1:1000) by theavidin-biotin-peroxidase complex (ABC) technique and incubationsequences as previously described (Kulaksiz et al., (2002) Proc. Natl.Acad. Sci. USA 99, 6796-6801; Iculaksiz et al., (2002) Am. J. Pathol.161, 655-664). The sections were incubated with the respectiveantibodies for 24 h at 4° C., followed by incubation with biotinylatedanti-rabbit IgG (Jackson Immunoresearch, West Grove, Pa., USA) for 30min diluted 1:200. The sections were then incubated for 30 min with apreformed complex of biotin-peroxidase/streptavidin (JacksonImmunoresearch), diluted in PBS (final concentrations:biotin-peroxidase, 0.7 μg/ml; streptavidin, 5 μg/ml). Theantigen-antibody binding sites were visualized by incubation of thesections in 07 mM diaminobenzidine hydrochloride/0.002% H202 in 0.05 MTris-HCl pH 7.6).

For immunofluorescence microscopy, tissue sections from human liver (2-4μm) were prepared with a cryotome (FrigoCut 2800E; Leica, Nussloch,Germany), air dried for 2 hours, and fixed for 10 min in cold acetone(−20° C.). Double-immunofluorescence labeling was performed as describedpreviously (Rost et al., (1999) Hepatology 29, 814-821) using thespecific hepcidin antibodies (diluted 1:1000) and monoclonal antibodyC219 (Id.) raised against canalicular P-glycoproteins (Centocor,Malvern, Pa.) diluted 1:30. After incubation with the respectiveantisera, staining was performed by incubation with Cy2-(1:200) andCy3-(1:600) labeled antibodies against mouse and rabbit IgG (Dianova,Hamburg, Germany). Micrographs were taken with an Olympus AX70microscope equipped with a digital camera (color view 12, soft imagingsystem SIS, Munster, Germany) and analysis software (SIS, Münster,Germany).

Specificity Controls

Method-dependent non-specificities were excluded by running controls asdescribed (Cetin et al., (1994), Proc. Natl. Acad. Sci. USA 91,2935-2939; Cetin et al., (1995) Proc. Natl. Acad. Sci. USA 92,5925-5929). Antibody specificities were tested by preadsorption of theantibodies with homologous and heterologous antigenic peptides (6.25 100μg/ml of the antiserum) (Kulaksiz et al., (2002) Proc. Natl. Acad. Sci.USA 99, 6796-6801; Kulaksiz et al., (2002) Am. J. Pathol. 161, 655-664).Preadsorption of the antibodies with homologous antigens atconcentrations as low as 6.25 μ/ml completely blocked immunostaining inthe liver tissues and cells, while preadsorption of the antibodies withheterologous antigens at concentrations up to 100 μg/ml had no effect onimmunostaining.

Hepcidin ELISA Competitive Binding Assay

Serum samples were obtained from 26 healthy individuals (13 women, 13men, aged 26-64, mean 43 yrs), from 35 patients with HH homozygous forthe C282Y mutation in HFE (14 women, 21 men, aged 23-82 years, mean 54years), with (15 patients) and without (20 patients) bloodlettingtherapy, and from 59 patients with renal insufficiency undergoingchronic hemodialysis (33 women, 26 men, aged 26-96 years, mean 57years). During sample collection it was taken care that the patients hadno infection. 19 patients of the group of renal insufficiency had renalanemia characterized by hemoglobin of maximum 11 g/dl. All patients withchronic renal insufficiency were treated 2-3 times a week with 3,000 IErecombinant human erythropoietin (EPO). 10 ml blood samples werewithdrawn into ice-chilled serum-tubes and centrifuged at 2,500×g for 10min at 4° C. Determinations were performed in duplicate using96-well-microtiter plates coated with 200 μl/well rabbit antihepcidinantibody EG(2)-HepN diluted 1:4000 in Tris buffered saline (TBS)containing 40 mM Tris-HCl (pH 7.3), 100 mM NaCl. 50 μl standardscontaining various amounts of synthetic peptides (0, 20, 100, 500, and1000 ng/ml) or human serum samples and 150 μl N-terminally biotinylatedhepcidin-(28-47) (Peptide Specialty Laboratories GmbH, Heidelberg,Germany) (2 ng/well) were added to each well and incubated for 1 hour atRT. After washing with TBST (TBS with 0.05% Tween 20), the biotinylatedantigen-antibody complexes were detected by streptavidin-peroxidaseenzyme (Dako, Hamburg, Germany) with the substrate tetramethylbenzidine(DRG Instruments GmbH, Marburg, Germany); the color reaction was stopedwith 1 M H₂SO₄ and the extinction of the solution was read at 450/630 nmwavelength.

Measured values of hepcidin in the four groups of interest were enteredin an EXCEL spreadsheet and evaluated using SAS WIN Version 8.2. Themeasured values were summarized by means of the following summarystatistics by diagnosis group: the number of observations, thearithmetic mean, standard deviation, minimum, median and maximum.Possible differences between groups were analyzed with pairwise WilcoxonU-tests. The level of significance was set to 5% (0.05). The correlationbetween prohepcidin and iron, ferritin or transferrin was analyzed bythe Spearman rank correlation.

Expression of Hepcidin and TfR2 in the Liver and HepG2 Cells

RT-PCR analysis demonstrated that hepcidin is expressed in human liver(Gehrke et al. (2003) Blood MS#2002-11-3610.R2). Similarly, a 192-bpexpected PCR product was detected in HepG2 cells (control), which werealready shown to express hepcidin (Pigeon C et al. (2001) J Biol Chem276, 7811-7819; Gehrke et al. 47 (2003 (FIG. 2, A). In addition, RT-PCRanalyses clearly revealed that TfR2 is expressed in the human liver andHepG2 cells (data not shown).

In Western blot analysis, all hepcidin antibodies [EG(1)-HepN,EG(2)-HepN, and EG(1)-HepC] coincidentally identified an immunoreactiveband of ˜10 kDa in extracts of human and guinea pig liver. This liverpeptide comigrated with an immunoreactive band recognized by thehepcidin antibodies in homogenates of HepG2 cells (FIG. 2, B-ID). Allantibodies also identified an immunoreactive protein-at ˜20 kDa in alllanes loaded with human and guinea pig liver extracts or HepG2 cellextracts. Western blot analysis of skeletal muscle extracts (control)showed neither the immunoreactive band of 10 kDa nor the band at 20 kDa(FIG. 2, B-D). Western blot analysis with TfR2 antibody BT-TFR21-Sresulted in a staining of an expected (Fleming et al., (2000) Proc.Natl. Acad. Sci. USA 97, 2214-2219)-105 kDa protein in extracts of mouseliver. In extracts of human liver and HepG 2 cells, a ˜95 kDaimmunoreactive TfR2 and to lesser extent a ˜105 kda immunoreactiveprotein was recognized by the same antibody (data not shown). Noimmunoreactivity was detected in the heart (control tissue).

Immunofluorescence in HepG2 Cells

Using epitope specific anti-hepcidin antibodies, expression of hepcidinpeptide in HepG2 cells was investigated by immunofluorescence analysis.All antibodies similarly identified hepcidin in HepG2 cells resulting ina strong immunoreactivity (FIG. 3). Coincident with the cellularlocalization of hepcidin, the TfR2 antibody detected TfR2 in the samecells (data not shown)

Cellular and Subcellular Localization of Hepcidin and TfR2.

Immunohistochemical studies with various region-specific antibodiesconsistently localized hepcidin to the hepatocytes in human liver (FIG.4). The Kupffer cells, endothelial cells, bile ducts, and the vascularsystem completely lacked hepcidin immunoreactivity. The same antibodiesdetected a strong hepcidinimmunoreactivity also in guinea pig liver(FIG. 4). Hepatic lobules were heterogeneous with respect to thehepcidin immunoreactivity: within a hepatic lobule, the hepcidinimmunoreactive cells were predominantly located in periportal zones, andthe frequency of hepcidin-positive cells continuously decreased from theportal triads toward the central veins (FIG. 5). Notably, distinctintercellular differences existed between the hepcidin positive cells:while most hepatocytes were strongly positive for hepcidin, othersdisplayed only a faint staining or were totally unreactive for hepcidin(FIG. 5). At the subcellular level, hepcidin immunoreactivity wasconfined to the basolateral (=sinusoidal) membrane domain of hepatocytesby immunohistochemistry; no immunoreactivity was found at the apicalmembrane domain of the respective cells (FIG. 2). Similarly,immunofluorescence analysis demonstrated a strong immunoreactivity forhepcidin at the basolateral membrane domain; immunoreactivity was absentfrom the apical membrane domain as revealed by double staining with theC219 antibody raised against canalicular P-glycoproteins (Rost et al.(1999) Hepatology 29, 814-821) (data not shown).

Corresponding to the localization of hepcidin, protein-specific antibodyBT-TFR21-S detected TfR2 in human and mouse liver. At the cellularlevel, TfR2 was found at the basolateral membrane of hepatocytes, whichrevealed distinct intercellular differences concerning the intensity ofimmunoreactivity (data not shown). Heterogeneity was also observedwithin a hepatic lobule with increasing immunoreactivity from thecentral veins to the portal triads.

Detection of Hepcidin Propeptide in Human Plasma

A stable pro-hepcidin ELISA assay (DRG Instruments GmbH, Marburg,Germany) with high reproducibility and sensitivity was developed withthe specific N-terminal hepcidin antibody EG(2)-HepN. As seen in FIG. 6,the ELISA revealed the highest resolving power between 4 and 400 ng/ml,a range, where pro-hepcidin concentrations in human serum weredetermined. As specificity control, the incubation in ELISA wasperformed with heterologous peptides. No crossreactivity was observedwhen heterologous peptides were used. The presence of pro-hepcidin inblood was verified by Western blotting analysis. All hepcidin antibodiesidentified in extracts of human serum a single hepcidin immunoreactiveband of ˜10 kDa molecular mass that comigrated exactly with theimmunoreactive hepcidin in liver tissues and HepG2 cell extracts (FIG.2, B-D)

ELISA Characteristics

The sensitivity of the assay was 3.95 ng/m]. There was no overlap withthe lowest standard (20 ng/ml). Serial dilutions of human pro-hepcidin,dissolved in the zero standard, run parallel to the standard curve ofthe pro-hepcidin ELISA with the range of recovery between 90.6-111.6%.Recovery expressed as percentage of observed from expected concentrationwas between 91.8 and 105.7%. Good precision was demonstrated (totalCV<10%) at three concentrations of pro-hepcidin tested across the assayrange.

Pro-Hepcidin Levels in Hereditary Hemochromatosis, Chronic RenalInsufficiency, and Renal Anemia

Using the sensitive hepcidin ELISA, pro-hepcidin in the range from51.6-153.4 ng/ml serum (mean±SE; 106.2±32.1 ng/ml) was detected in thehealthy control group of 26 volunteers (table 1). In patients with HH,the concentrations of pro-hepcidin were 12.1 to 153.9 ng/ml serum(mean±SE; 70.2±38.1 ng/ml). These concentrations were significantlylower compared to that in control subjects (P<0.05) (table 1). Thepro-hepcidin concentrations varied from 31.1 to 471.3 ng/ml (mean±SE;1481±88.0 ng/ml) in serum of patients suffering from CRI and weresignificantly increased compared with that in control subjects (P<0.01)and HH(P<0.001). In contrast, pro-hepcidin levels in hemodialysispatients with RA were significantly decreased (115.0±53.1 ng/ml; range,20.5-252.4 ng/ml) (P=0.05) compared to patients with CRI (table 1)

No significant correlation was found in our samples (serum from HH, CRI,and RA) between pro-hepcidin and iron, ferritin or transferrinsaturation. The test of difference from zero showed no significance.

TABLE 1 RESULTS OF PAIRWISE U-TESTS (P-VALUE) Chronic Renal RenalInsufficiency Anemia Hemochromatosis Control 0.0419 0.6131 <0.0005Chronic Renal 0.23   <000l Insufficiency Renal Anemia  0.002Discussion

RT-PCR analyses with specific primers showed that hepcidin is highlyexpressed in HepG2 cells (control), a well-differentiated hepatocellularcarcinoma cell line (Aden et al. (1979) Nature 282, 615-616)demonstrating in many aspects the physiology of normal hepatocytes.Using the appropriate primer specifications and combinationssuccessfully employed in HepG2 cells, the RT-PCR studies confirmedexpression of hepcidin in the human liver. Three different antibodiesrecognizing different epitopes in the hepcidin precursor molecule(FIG. 1) concurrently identified an immunoreactive peptide of ˜10 kDa byWestern blot analysis not only in HepG2 cells but also in liver extractsof two species, man and guinea pig. The apparent molecular mass of thisimmunoreactive peptide is in accordance with the predicted molecularmass deduced for the hepcidin prohormone from the cDNA sequence (PigeonC et al. (2001) J Bid Chem 276, 7811-7819) (FIG. 1). Interestingly, asecond immunoreactive band of ˜20 kDa was detected by all hepcidinantibodies in extracts of HepG2 cells and human and guinea pig liver butwas lacking in the control tissue. This immunoreactive protein mayreflect a dimeric type of hepcidin. In fact, in a previous study anaggregation property and a possible formation of multimers weredescribed for hepcidin-25 but not for hepcidin-20 (Hunter et al. (2002)J Bid Chem 277, 37597-37603).

Immunocytochemical studies with the region- and moleculardomain-specific hepcidin antibodies revealed a strong immunoreactivityin HepG2 cells demonstrating expression of hepcidin in these cells asalready shown by molecular biological techniques (Gehrke et al. (2003)Blood MS#2002-11-3610.R2). Immunohistochemical and immunofluorescenceinvestigations with these different hepcidin antibodies indicated that,in human and guinea pig liver, hepcidin is specifically localized inhepatocytes mainly located around the portal triads. The coincidentstaining by different region-specific antibodies not only in the humanand guinea pig liver but also in the HepG2 cells points to hepatocytesbeing the source of hepcidin. Hepcidin immunoreactivity decreased fromthe periportal zones towards the central veins. This zonation within theportal lobules may have a functional significance, since the periportalhepatocytes have first-pass access to portal veins bringing iron-richblood from the gut. Notably, distinct intercellular differences existedbetween the hepcidin-positive cells with respect to the density ofhepcidin immunoreactivity that may reflect intercellular differences inexpression or secretion of hepcidin.

At the subcellular level, hepcidin was concentrated at the basolateralmembrane domain of hepatocytes. No immunoreactivity was found at theapical membrane domain. The discrete distribution pattern of hepcidin atthe subcellular level may infer a basolaterally directed release ofhepcidin into the liver sinusoids. This directional secretion route isadditionally substantiated by the detection of hepcidin prohormone(FIG. 1) in human serum (see below); consequently, these findingsprovide further evidence that hepatocytes may regulate iron metabolismin an endocrine fashion via the secretion of prohepcidin.

To analyze the expression and cellular distribution of TfR2 as well asthe respective target membrane domains, RT-PCR, Western blot andimmunohistochemical studies at the cellular level were performed. Asshown in previous studies RT-PCR analyses revealed that TfR2 is highlyexpressed in human liver. (Fleming et al., (2000) Proc. Natl. Acadi.Sci. USA 97, 2214-2219). The presence of this protein was confirmed byWestern blot studies using BT-TFR21-S antibody specific to human andmouse TfR2. A ˜105 kDa immunoreactive protein was detected in mouseliver extracts; this molecular mass of immunoreactive TfR2 is slightlylarger than the expected 95 kDa (Fleming et al., (2000) Proc. Natl.Acadi. Sci. USA 97, 2214-2219) and may represent some posttranslationalmodifications as described previously (Kawabata et al., (2000) J. Biol.Chem. 275, 16618-16625). Under identical conditions, however, theTfR2-antibody identified the protein at the expected 95 kDa molecularmass and with a lower affinity the 105 kDa protein in human liverextracts. The discrepancy between the immunoblots of human and mouseliver may be due to interspecies differences.

Immunohistochemical investigations revealed that TfR2 is localized tohepatocytes of human and mouse liver; coincident with the cellulardistribution of hepcidin, the protein-specific antibody localized TfR2exclusively at the basolateral membrane. This type of membrane-specificassociation of TfR2 argues particularly for a basolateral activation ofTfR2, which is involved in iron metabolism by binding diferrictransferrin and mediating uptake of transferrin-bound iron from theblood into hepatocytes (Philpott, C. C. (2002) Hepatology 35, 993-1001;Subramaniam et al., (2002) Cell Biochem. Biophys. 36, 235-239). Notably,a similar lobular zonation as described for hepcidin was observed forTfR2 with decreasing immunoreactivity from the periportal zones towardthe central veins.

Since an interaction between hepcidin and TfR2 at the cellular level hasbeen discussed in previous studies (Nicolas et al., (2001) Proc. Natl.Acad. Sd. USA 98, 8780-8785; Frazer et al., (2002) Gastroenterology 123,835-844), the coexistence of hepcidin and TfR2 in HepG2 cells awell-differentiated hepatocellular carcinoma cell line (Aden et al.,(1979) Nature 282, 615-616) was analyzed, demonstrating in many aspectsthe physiology of normal hepatocytes. RT-PCR studies using theappropriate primer specifications and combinations successfully employedin the human liver identified expression of hepcidin and TfR2 in HepG2cells. At the translational level, the presence of hepcidin and TfR2 inHepG2 cells was confirmed by Western blot studies that yieldedimmunoreactive protein bands of correct molecular weights, comigratingwith the corresponding immunoreactive bands from the liver tissues. Theco-localization of the respective proteins in HepG2 cells wasparticularly substantiated by immunocytochemistry using thecorresponding region- and molecular domain-specific antibodies. Allantibodies demonstrated hepcidin-labeling in HepG2 cells, revealing agranular immunoreactivity pattern in these cells that inferslocalization of the peptide to small secretory vesicles, alreadydemonstrated in hepatocytes by electron microscopy (Schwartz et al.,(1985) EMBO J. 4, 899-904). TfR2 was immunocytochemically localized,with a peculiar distribution pattern, to HepG2 cells.

On the basis of present data at the transcriptional and translationallevel, hepcidin and TfR2 are coexpressed in the liver and colocalized atthe basolateral membrane domain of hepatocytes. In addition to acoincident localization of TfR2 and hepcidin at the cellular level, asimilar distribution of these molecules within the hepatic lobules witha concentrated immunoreactivity in periportal zones and a decreasingstraining toward the central veins was also detected. The coordinateexpression of these proteins in a common (basolateral) membrane domainand their similar lobular zonation argue for a morphofunctional couplingof the regulating peptide hormonohepcidin and the transferrin-bound ironuptake via TfR2. Indeed, different data substantiate the interactionbetween hepcidin and TfK2. First, alterations in transferrin saturation,probably sensed by TfR2, modulate the expression of hepatic hepcidin(Philpott, C. C. (2002) Hepatology 35, 993-1001). Second, as revealedfrom quantitative RT-PCR analyses on human liver, hepatic expression ofTfR2 correlates significantly with hepcidin expression regulated by thetransferrin saturation (S. G. Gehrke, H. Kulaksiz et al. unpublisheddata). Third, hepcidin and TfR2 are colocalized at a common cellmembrane domain and reveal the same lobular distribution with a strongimmunoreactivity in periportal zones, the site, where in case ofmutations that abrogate expression of TfR2 (Fleming et al., (2002) Proc.Natl. Acad. Sci. USA 99, 10653-10658) and hepcidin (Nicolas et al.,(2001) Proc. Natl. Acad. Sci. USA 98, 8780-8785) but also hepcidin (Zhouet al., (1998) Proc. Natl. Acad. Sd. USA 95, 2492-2497; Levy et al.,(1999) Blood 94, 9-11) and B2m (Santos et al., (1996) J. EZp. Med. 184,1975-1985) hepatic iron overloading occurs. Fourth, mutations in theTfR2 gene were reported to lead to hemoechromatosis (Camasehella et al.,(2000) Nat. Genet. 25, 14-15); this may result from decreased hepcidinexpression, which, in turn, results in increased iron absorption(Nicolas et al., (2001) Proc. Natl. Acad. Sd. USA 98, 8780-8785).

The simultaneous existence of hepcidin and TfR2 in HepG2 cells and theircommon polarized localization and lobular distribution in the liver mayindicate that hepcidin is an intrinsic hepatic peptidemorphofunctionally coupled to TfR2, which is regulated by transferrinsaturation and, in turn, modulates expression of hepcidin. Hence,pertinent findings are expected from studies on the signaling pathway ofhepcidin.

Since blood-forming tissues and sites of iron storage, such as theliver, transmit signals to the intestinal cells that indicate the body'srequirements for dietary iron (Philpott C. C. (2002) Hepatology 35,993-1001), hepcidin is a candidate signaling factor secreted from thehepatocytes and regulating the intestinal iron absorption. However,prior to the present invention, there was controversy about theexistence of certain molecular forms of hepcidin in the blood (Krause etal. (2000) FEBS Lett 489, 147-150; Park et al. (2001) J Biol Chem 276,7806-7810; Hunter et al. (2002) J Biol Chem 277, 37597-37603).

To analyze whether the prohormone of hepcidin is secreted into theblood, and to assess the range of prohepcidin levels in human serum ofhealthy volunteers and of patients with different diseases, an ELISA wasdeveloped by applying the N-terminal antibody EG(2)-HepN raised againsthepcidin prohormone. Although the C-terminal antibody EG(1)-HepCrevealed specific results in dot blot (data not shown), Western blot,immunohistochemistry, and immunofluorescence experiments (FIGS. 1-4), noimmunoreactivity could be obtained in ELISA. The compact folding patternof hepcidin and its tertiary structure in the blood may account for theinability of the EG(1)-HepC antibody to identify circulating hepcidin.

The ELISA with antibody EG(2)-HepN was characterized by a highreproducibility, stability and sensitivity with a detection limit of3.95 ng/well and a powerful resolution in the range of 4 to 400 ng/ml;the range, where hepcidin concentrations were determined. In human serumfrom healthy individuals (n=26), prohepcidin was measured in the rangefrom 51.6 to 153.4 ng/ml (mean±SE; 106.2±32.1 ng/m].), which iscomparable with the concentration of known regulating peptide hormonesand approximately 11-fold higher than the concentration of hepcidin inhuman urine (Park et al. (2001) J Biol Chem 276, 7806-7810).Interestingly, the measured concentrations exhibited a wide range ofprohepcidin indicating that the peptide may be subject to strongregulation.

The cDNA structure suggests that hepcidin is translated as an 84 aaprepropeptide that is N-terminally processed to the 20-25 amino acidpeptides (Park et al. (2001) (FIGS. 1 and 7). Although a strongconsensus sequence for a signal sequence cleavage site is locatedbetween Gly²⁴ and Ser²⁵ that would result in a 60 residue propeptide,previous studies failed to isolate the larger propeptide from nativesources like liver tissue and blood (Park et al. (2001)). In addition totechnical difficulties, the abundance of propeptide convertases in theliver may inhibit the isolation of certain propeptides. In this context,recent studies have shown that the human circulating form of hepcidin,described by two research groups in blood (Krause et al. (2000) FEBSLett 489, 147-150) and in urine (Park et al. (2001) J Biol Chem 276,7806-7810), consists of the C-terminal 20-25 amino acids of the protein.However, ELISA measurements of the present invention were performed withthe specific-antibody raised against the N-terminus of hepcidinprecurcor, implying that besides the 20-25 amino acid processed formsthe hepcidin prohormone is secreted and circulates in human blood.Indeed, the potential release of prohepcidin into the blood wasconfirmed by Western blot analysis. All hepcidin antibodies identifiedin extracts of human serum a single hepcidin band of ˜10 kDa thatcomigrated exactly with the immunoreactive hepcidin in tissue extractsof the liver and HepG2 cells (positive control; FIG. 1). Hepcidinfragments smaller than 10 kDa were not detected. The presence ofprohepcidin in the human serum indicates that hepatocytes secrete theprohormone of hepcidin that may decrease dietary iron absorption via anendocrine pathway.

To analyze the significance of hepcidin in patients with iron overload,the present invention provides hepcidin concentrations in serum of 35 HHpatients homozygous for C282Y mutation in HFE with the typicalcharacteristics of iron overload detected in all HH patients understudy. Hepcidin concentrations were not increased in these individualsto reduce the intestinal iron absorption as supposed previously (Flemingand Sly (2001) Proc Natl Acad Sci USA 98, 8160-8162). Prohepcidin levelsin serum of HH patients were unexpectedly downregulated, not only inuntreated patients, but also in individuals undergoing a weeklybloodletting therapy. Compared to healthy volunteers, the prohepcidinconcentrations were markedly reduced from 106.2 to 70.2 ng/ml serum. Nodifference was observed between treated and untreated HH patients. Thesefindings are in line with previous HH studies showing that the liverhepcidin expression is significantly decreased in the hfe knockout mouse(Ahmad et al. (2002) Blood Cells Mol Dis 29, 361-366; Muckenthaler etal. (2003) Nat Genet 34, 102-107) and in patients with HFE-associatedhemochromatosis. They are also in accordance with in vitro studiesdemonstrating that iron loading of primary human hepatocytes and HepG2cells downregulate hepcidin mRNA (Gehrke et al. (2003) BloodMS#2002-11-3610.R2; Nemeth et al. (2003) Blood 101, 2461-2463). Sinceiron absorption is enhanced in HH despite iron overload (Pietrangelo A.(2002) Am J Physiol Gastrointest Liver Physiol 282, G403-414; PhilpottC. C. (2002) Hepatology 35, 993-1001; Anderson and Powell (2002) Int JHematol 76, 203-207), and constitutive hepcidin expression preventediron overload in a mouse model of hemochromatosis (Nicolas et al. (2003)Nat Genet 34, 97-101), it is assumed that the hepcidin regulation isdisrupted in HH patients. The depressed concentrations of hepcidin areobviously not able to inhibit sufficiently the elevated intestinal ironabsorption. Moreover, based on the findings that the liver hepcidinexpression is significantly decreased in the hfe knockout mouse (Ahmadet al. (2002) Blood Cells Mol Dis 29, 361-366; Muckenthaler et al.(2003) Nat Genet 34, 102-107) and in HH patients (Bridle et al. (2003)Lancet 361, 669-673), the lack of prohepcidin upregulation in HH despiteiron overload indicates that HFE may be involved in the regulation ofserum hepcidin levels.

Although previous studies have demonstrated that urinary hepcidinexcretion correlates well with serum ferritin concentrations (Nemeth etal. (2003) Blood 101, 2461-2463), in the present study, no correlationwas found between the circulating prohepcidin and the serum iron orferritin levels in HH or dialysis patients. Likewise, no correlation wasdetected between prohepcidin and the transferrin saturation, which issupposed to regulate the expression of liver hepcidin (Gehrke et al.(2003)), although the HH patients under study were not affected byanemia, hypoxia or inflammation, representing hepcidin influencingparameters. The data suggests that the regulation of prohepcidin levelsin the serum by iron stores involves complex indirect effects (Nemeth etal. (2003) Blood 101, 2461-2463).

Since hepcidin has also been isolated from urine, the present inventionprovides for the evaluation of hepcidin regulation in renal insufficientpatients. In contrast to HH patients and to healthy subjects,concentrations of immunoreactive prohepcidin in the serum of patientswith CRI were significantly increased from 106.2 ng/ml in healthysubjects to 148.1 ng/ml. Enhanced levels of prohepcidin in dialysispatients suggests that the kidneys may be involved in the metabolismand/or elimination of the circulating peptide. However, it is presentlyunclear if the urinary hepcidin is only filtered from the blood ororiginates from the kidney. Based on the present invention, it cannot beexcluded that hepcidin is released at least partly from the kidneys,since it was also found in renal tubular cells (Kulaksiz et al. (2003)unpublished data).

The present invention provides for a determination of prohepcidin serumlevels in dialysis patients with RA, a well recognized complication ofprogressive renal failure, which is characterized by normochromic,normocytic erythrocytes. In comparison to healthy subjects, theimmunoreactive prohepcidin concentrations were not significantly higherin patients with RA (mean, 115.0 ng/ml). Despite the terminal renalinsufficiency in these patients leading to accumulation of the peptidehormone, the prohepcidin levels were significantly lower than indialysis patients without anemia (mean, 148.1 ng/ml). From the presentinvention it is concluded that hepcidin regulation in RA is differentthan that in anemia of inflammation or of hepatic adenomas.Downregulation of prohepcidin in RA reflects a reactive physiologicalmodulation of the peptide to enhance intestinal iron absorption and ironrelease from reticuloendothelial macrophages. The present inventionprovides that prohepcidin is increased in the group of patients with CRIwithout anemia despite EPO therapy. Thus, it is concluded that hepcidinis decreased in RA because of blood loss, which may be a reason forhepcidin downregulation (Nicolas et al. (2002) J Clin Invest 110,1037-1044).

The present invention provides an ELISA to measure prohepcidin levels inhuman serum. This assay is non-invasive and easy to perform, thusappropriate for routine work. The prohepcidin assay is due to itsprecision, sensitivity, reproducibility and exact determination ofhepcidin-(28-47) of human serum samples. Application of the presentELISA allows for the first time the detection and determination ofprohepcidin in patients suffering from several disorders of ironmetabolism. Further studies are required to identify the exact molecularmechanism of prohepcidin action in various iron states. The presentinvention also provides that hepcidin agonists and antagonists should bepotential drugs in the prevention and treatment of iron disorders.

To understand the role of hepcidin, knowledge about the cellular originand the signaling pathway of the peptide is necessary. In this respect,the present invention describes hepcidin immunoreactivity in human andguinea pig liver, where it is localized to the basolateral membranedomain of hepatocytes. Previous studies have speculated on a possibleconnection between these cells and the absorptive enterocytes (Hunter etal., (2002) J. Biol. Chem., M205305200; Anderson et al., (2002) Biochem.Soc. Trans. 30, 724-726). The present invention describes the detectionof prohepcidin in the human plasma thereby indicating that hepatocytessecrete the prohormone of hepcidin that may decrease dietary ironabsorption via an endocrine pathway. Moreover, hepcidin was detected inHepG2 cells, where the newly discovered transferrin receptor type 2 wasalso found (data not shown).

Enzyme Immunoassay for the Quantitative Measurement of Hepcidin in Humanor Animal Serum and Other Body Fluids.

In one embodiment of the invention a Hepcidin enzyme immunoassay (“EIA”)is used. An EIA is a solid phase enzyme-linked immunosorbent assay(ELISA) based on the competitive principle. Microtiter wells of a 96well microtiter plate are coated with a polyclonal rabbit anti-hepcidinantibody directed against Hepcidin-(28-47). An unknown amount ofProhepcidin present in the sample and a fixed amount of Hepcidin-(28-47)conjugated with a biotin molecule compete for the binding sites of theHepcidin antibodies immobilized on the wells. After one hour incubationthe microtiter plate is washed to stop the competition reaction. In thefollowing incubation the bound biotin molecules are detected withstreptavidin horseradish peroxidase. After one half hour of incubationthe plate is washed a second time. Having added the substrate solutionthe concentration of Hepcidin is inversely proportional to the opticaldensity measured.

Materials:

Microtiter wells; wells coated with Anti-Hepcidin antibody (96 wells);Reagent: Biotin Conjugate (Hepcidin conjugated to biotin) 7 ml.;Reference Standard Set, 1.0 ml each; 0, 20, 100, 500, 1000, 2000 ng/ml;Prohepcidin Controls, low and high, 2 vials (lyophylizate); Reagent:Enzyme Complex (Streptavidin conjugated to horseradish peroxidase(“HRP”)) 14 ml; Reagent: Substrate Solution-HS-TMB, 14 ml; StopSolution, 0.5M H₂SO₄, 14 ml; Wash Solution, 40×, 30 ml; Amicrotiterplate reader (450±10 nm) (e.g., the DRG InstrumentsMicrotiterplate Reader); Precision micropipettes with disposable tipsfor 50 and 100 μl; Standard refrigerator; Absorbent paper; Deionizedwater.

While this embodiment has been described in terms of preferredmaterials, a person skilled in the art of the invention will appreciatethat other materials can be used in the invention. For example, one ofskill in the art will appreciate that complementary binding moietiesother than biotin/streptavidin, as well as enzyme/substrate combinationsother than horse radish peroxidase/peroxide, may be used in theinvention.

Storage Conditions.

When stored at 2° to 8° C. unbroken reagents will retain reactivityuntil expiration date. Do not use reagents beyond this date. Microtiterwells must be stored at 2° to 8° C. Once the foilbag has been brokencare should be taken to close it tightly again. The immuno-reactivity ofthe coated microtiter wells is stable for approx. 6 weeks in the broken,but tightly closed plastic zip pouch containing the desiccant.

Specimen Collection and Preparation.

Human or animal serum or EDTA plasma should be used in the assay. Nospecial pretreatment of the biological sample is necessary. Thebiological sample may be stored at 2-8° C. for up to 24 hours, andshould be frozen at −20° C. or lower for longer periods. Do not usegrossly hemolyzed or grossly lipemic specimens. For other samplematerial a special extraction protocol may be necessary.

Performance of the Assay: General Remarks:

All reagents and specimens must be allowed to come to room temperaturebefore use. All reagents must be mixed without foaming.

Once the test has been started, all steps should be completed withoutinterruption.

Use new disposable plastic pipette tips for each reagent, standard orspecimen in order to avoid cross contamination. For the dispensing ofthe Substrate Solution and the Stop Solution avoid pipettes with metalparts.

Pipette standards and samples onto the bottom of the well. For pipettingof Enzyme Conjugate and Stop Solution it is recommended to hold thepipette in a vertical position above the well and dispense thecorrespondent solution into the center of the well so that a completemixing of Enzyme Conjugate with sample or standard and of the StopSolution with the Substrate Solution is achieved.

Before starting the assay, it is recommended that all reagents be ready,caps removed, all needed wells secured in holder, etc. This will ensureequal elapsed time for each pipetting step without interruption.

As a general rule the enzymatic reaction is linearly proportional totime and temperature. This makes interpolation possible for fixedphysico-chemical conditions. If in a test run the absorbance of ZeroStandard is lower than 1.0 or above the upper performance limit of yourmicrotiterplate spectrophotometer you can extend or reduce theincubation time of the final enzymatic formation of color to 30 or 10minutes accordingly. Since calibrators are assayed in each run,absorbance fluctuations do not affect the result.

The Substrate Solution should be colorless or slightly blue or green. Ifthe solution is dark blue the reagent is unusable and must be discarded.

During incubation with Substrate Solution avoid direct sunlight on themicrotiter plate.

Reagent Preparation.

Reference Standards and Controls: Reconstitute the lyophilized contentsof the standard/control vials with 1.0 ml bidistilled Water. Note: Thereconstituted standards/controls are stable for 6 days at 2-8° C. Forlonger storage freeze at −20° C. Wash Solution: Add deionized water tothe 40× concentrated Wash Solution (contents: 30 ml) to a final volumeof 1200 ml. The diluted Wash Solution is stable for 2 weeks at roomtemperature.

Assay Procedure.

Secure the desired number of coated strips in the holder.

Dispense 50 μl of Hepcidin Standards into appropriate wells.

Dispense 50 μl of sample into selected wells.

Dispense 50 μl of Biotin Conjugate into each well.

Thoroughly mix the plate for 10 seconds. It is important to havecomplete mixing in this step.

Incubate for 60 minutes at room temperature.

Briskly shake out the contents of the wells.

Rinse the wells 3 times with diluted Wash Solution (400 μl per well).Strike the wells sharply on absorbent paper to remove residual droplets.

Add 100 μl Streptavidin HRP Complex to all wells.

Incubate for 30 minutes at room temperature.

Briskly shake out the contents of the wells.

Rinse the wells 3 times with diluted Wash Solution (400 μl per well).Strike the wells sharply on absorbent paper to remove residual droplets.

Add 100 μl of Substrate Solution to each well, at timed intervals.

Incubate for 15 minutes at room temperature.

Stop the enzymatic reaction by adding 100 μl of Stop Solution to eachwell at the same timed intervals as in step 10 and determine theabsorbance of each well at 450±10 nm.

Final Reaction Stability.

It is recommended that the wells be read within 30 minutes followingstep 15.

Calculation of Results.

Any microwell reader capable of determining the absorbance at 450±10 nmmay be used. The Testosterone value of each sample is obtained asfollows:

-   -   a. Using linear-linear or semi log graph paper, construct an        standard curve by plotting the average absorbance (Y) of each        Reference Standard against its corresponding concentration (X)        in ng/ml. For construction of the standard curve we recommend a        four parameter logistic function.    -   b. Use the average absorbance of each sample to determine the        corresponding Testosterone value by simple interpolation from        this standard curve, multiplying by the initial sample dilution,        if necessary.

A DRG ELIZA MAT 3000 and the DRG Regression Program allow the readingand computer assisted interpretation using a four parameter logisticfunction.

Example of a Standard Curve.

The following data is for demonstration only and cannot be used in placeof data generations at the time of assay.

Standard OD at 450 nm Standard 0 (0 ng/ml) 1.79 Standard 1 (20 ng/ml)1.67 Standard 2 (100 ng/ml) 1.33 Standard 3 (500 ng/ml) 0.82 Standard 4(1000 ng/ml) 0.61 Standard 5 (2000 ng/ml) 0.43Performance Characteristics Sensitivity.

FIG. 6. illustrates a representative ELISA for circulating humanprohepcidin standard curve with concentrations of hepcidin(28-47) inng/ml and the extinction of the ELISA solution at 450 nm wavelength.

TABLE 2 PROCEDURE FLOW SHEET DRG HEPCIDIN ELISA KIT Standard/ Biotin-Streptavidin Substrate Stop Sample Conjugate HRP Complex SolutionSolution Description μl μl μl μl μl Standard 0 50 50 Mix for 100Incubate 100 Incubate 100 Read the 10 for 30 for 15 OD at seconds.minutes minutes 450 nm Incubate at room at room with a for 60temperature. temperature Microtiter- minutes Rinse plate at room thewells reader. Standard 1 50 50 temperature 100 3 times 100 100 Standard2 50 50 Rinse 100 with 100 100 the wells diluted Standard 3 50 50 3times 100 Wash 100 100 Standard 4 50 50 with 100 Solution 100 100Standard 5 50 50 diluted 100 400 100 100 Standard 6 50 Wash 100 μl/well100 100 Solution Sample 1 50 50 400 100 100 100 Sample 2 50 50 μl/well.100 100 100 Sample 3 50 50 100 100 100 Sample 4 50 50 100 100 100 Sample5 50 50 100 100 100

The analytical sensitivity was calculated from the mean minus 2 SD(SD=0.055) of 21 replicates (n=21) analysis of zero standard.

The sensitivity of the assay is 3.95 ng/ml. The linearity of the assaywas evaluated by diluting of the samples (serum) having differentHepcidin levels with zero standard. The Hepcidin content in the dilutedsamples was assayed by the ELISA. Three dilutions were performed foreach sample and the percentage recovery rates were calculated.

Mean value (ng/ml) 591.6 157.5 179.4 Average % Recovery 99.1 107.5 104.6Range of % Recovery 90.6-108.2 106.3-107.2 92.3-111.6

The analytical recovery of Hepcidin was estimated at 3 differentconcentrations in serum samples. Increasing amounts of unlabeledHepcidin (50 ng/ml, 250 ng/ml, 500 ng/ml) were added to the samples withvarious initial Hepcidin concentrations. Each sample (non spiked andspiked) was assayed. The Hepcidin concentrations were measured and thepercentage recovery rates were calculated.

Mean value (ng/ml) 273.8 116.8 82.3 Average % Recovery 93.1 94.7 97.1Range of % Recovery 91.8-94.3 89.2-98.7 94.5-105.7

The intra-assay precision (within-run) variation was determined byrepeated measurements (n=12) of 3 control samples with differentHepcidin contents.

Sample 1: mean=426.7; SD=20.2; CV (%)=4.69

Sample 2: mean=210.7; SD=8.58; CV (%)=4.07

Sample 3: mean=110.7; SD=4.74; CV (%)=4.28

The inter-assay precision (between-run) variation was determined byrepeated (n=23) measurement (3×) of 3 different control samples in threedifferent kit lots.

Sample 1: mean=431.96; SD=20.8; CV (%)=4.82

Sample 2: mean=216.17; SD=14.44; CV (%)=6.68

Sample 3: mean=109.8; SD=10.72; CV (%)=9.76.

Example 2 Expression of Hepcidin in the Human Kidney Hepcidin isExpressed in Distal Renal Tubuli and Released into the Urine

It is widely believed that iron homeostasis is mainly controlled in thegastrointestinal tract by absorption of dietary iron. However, recentstudies show that the kidneys are also involved in iron metabolism.Since the iron regulatory and antimicrobial peptide hepcidin wasoriginally isolated from human urine, Applicants investigated thecellular and subcellular localization of hepcidin in the mammaliankidney and developed an ELISA assay to analyze pro-hepcidinconcentrations in serum and urine.

The expression and cellular localization of hepcidin was shown byRT-PCR, Western blot, and immunocytochemistry in human, mouse, and ratkidney with hepcidin specific polyclonal antisera. Its serum and urineconcentrations were determined by a sensitive ELISA.

Hepcidin is expressed in human, mouse, and rat kidney. Western blotanalysis with region-specific antisera identified a ˜9.5 kDa peptidecorresponding to the apparent molecular mass of prohepcidin.Localization studies revealed that hepcidin is expressed in the distaltubuli of renal cortex and renal outer medulla. At the subcellularlevel, hepcidin is localized to the apical membrane domain of secretorytubuli cells which, based on its additional presence in the urine, isobviously released apically into the urine. Enhanced levels ofpro-hepcidin (156.8 ng/ml, healthy volunteers 104.2 ng/ml) weredetermined in patients with CRI indicating that kidneys may metabolizeand/or eliminate the circulating hormone.

From the expression of hepcidin in the mammalian kidney, Applicantsconclude that the iron regulatory hormone hepcidin is an intrinsic renalpeptide, which is not only eliminated/metabolized by the kidney, butalso synthesized in the kidney tubule system and released luminally intothe urine. Localization of hepcidin in the kidney implicates aregulatory role for this peptide in the renal tubule system.

Introduction

Recent studies have found abnormal hepcidin expression (Muckenthaler etal., (2003) Nat Genet, 34:102-107) and disrupted hepcidin regulation(Bridle et al., (2003) Lancet, 361:669-673; and Kulaksiz et al., (2003)Gut, in press) in HFE-associated hemochromatosis and association ofhepcidin mutations with severe juvenile hemochromatosis (Roetto et al.,(2003) Nat Genet, 33:21-22). Based on these observations, it has beensuggested that hepcidin is a key component of iron homeostasis that actas a negative regulator of iron absorption in the small intestine andiron release from macrophages (Nicolas et al. (2002) Proc Natl Acad SciUSA 99, 4596-4601).

While the majority of studies concentrate on the regulation and functionof hepcidin in the liver, the major site of hepcidin production (Park etal.; Kulaksiz et al., (2003) Gut, in press), indications accumulate thatthis peptide may also play a role in the kidney and urinary tract (Id.,Wareing et al., (2003) Am J Physiol Renal Physiol, Epub ahead of print;and Ferguson et al., (2003) Kidney Int, 64:1755-1764). It is widelybelieved that iron homeostasis is mainly controlled in thegastrointestinal tract at the level of uptake from the diet. The currentdogma is that there is no excretory route for iron in the organism.However, recent studies have shown that the kidney plays an importantrole in iron homeostasis (Wareing et al., (2003) Am J Physiol RenalPhysiol, Epub ahead of print; Ferguson et al., (2003) Kidney Int,64:1755-1764; and Gunshin et al., (1997) Nature, 388:482-488); asignificant proportion of iron in serum is available for ultrafiltrationby the glomerulus and the majority of iron filtered at the glomerulus isreabsorbed (Wareing et al., (2000) J Physiol, 524.2:581-586).

Hence, it is reasonable to analyze whether hepcidin is also present inthe kidney as a local peptide. Therefore, Applicants have raisedantisera against various epitopes of the hepcidin precursor molecule andinvestigated three mammalian species at transcriptional andtranslational level. Our findings indicate that, beside the eliminationof serum hepcidin in the kidney, the peptide is also produced as anintrinsic hormone in the distal tubuli cells of the mammalian kidney andreleased luminally into the urine, implicating a regulatory role forhepcidin in the kidney and/or urinary tract.

Materials and Methods

TISSUES AND TISSUE PREPARATION: Human kidney samples (n=5) used in thepresent study were obtained after kidney resection in adult patientswith hypernephroma. Human liver samples (n=7) used in the present studywere obtained after hemi-hepatectomy in adult patients with livermetastases (Kulaksiz et al., (2003) Gut, in press). Healthy tissues werefixed in 4% paraformaldehyde or in Bouin's fixative forimmunohistochemistry or immediately frozen in liquid nitrogen for RT-PCRand Western blot. Rats (n=5) and mice (n=5) were anesthetized andsubsequently sacrificed by cervical dislocation. Tissue specimens fromkidney and liver were resected and immediately frozen in liquid nitrogenfor RT-PCR or Western blot analysis or fixed in paraformaldehyde.

PEPTIDE SYNTHESIS, IMMUNIZATION PROCEDURE, AND ANTIBODIES: From thepublished pro-hepcidin sequence (Krause et al., (2000) FEBS Lett,480:147-150; Pigeon et al., (2001) J Biol Chem, 276:7811-7819), thepeptides hepcidin-(28-47) and hepcidin-(70-84) were synthesized as Cterminal amides using a standard Fmoc protocol (Kulaksiz et al., (2002)Proc Natl Acad Sci USA, 99:6796-6801; and Kulaksiz et al., (2002) Am JPathol, 161:655-664). Peptides were coupled to keyhole limpet hemocyaninusing m-maleimidobenzoyl-N-hydroxysuccinimide ester, and two SPF rabbits(Charles River-If fa Credo) were immunized with each peptide conjugate(Eurogentec, Seraing, Belgium). The antibodies EG(1)-HepC, EG(2)-HepC[each directed against prohepcidin-(70-84)], and EG(1)-HepN andEG(2)-HepN [each directed against prohepcidin-(28-47)] have beengenerated, characterized, and used (Kulaksiz et al., (2003) Gut, inpress).

EXPRESSION ANALYSES IN THE KIDNEY: Based on the GenBank cDNA sequences,the following primers were constructed and used: human hepcidin(database accession number NM021175) given in 5′-3′ orientation, 5′-CTGCAA CCC CAG GAC AGA G-3′ and 5′-GGA ATA AAT AAG GA GGG AGG GG-3′; rathepcidin (# NM 053469), 5′-ACA GAA GGC AAG ATG GCA CT-3′ and 5′-GAA GTTGGT GTC TCG CTT CC-3′, mouse hepcidin1 (# NM 032541), 5′-CGA TAC CAA TGCAGA AGA GAA GG-3′ and 5′-TTC AAG GTC ATT GGT GGG GA-3′. The primersdisplayed no homology to any previous reported sequences.

RNA isolation was performed using Qiagen RNAeasy kit including DNAdigestion. Reverse transcription (RT)-PCR analysis was performed asdescribed previously (Kulaksiz et al., (2002) Proc Natl Acad Sci USA,99:6796-6801; and Kulaksiz et al., (2002) Am J Pathol, 161:655-664).After an initial denaturation of 94° C. for 4 min, reactions weresubjected to 30 cycles of the following thermal program: 94° C. for 30s, 60° C. for 30 s, and 72° C. for 30 5; this program was followed by afinal 5-mm elongation step at 72° C. Amplification products were run onan ethidium bromide-stained 1.8% 89 mE Tris/89 mE boric acid/2 mM EDTA(pH 8.3) agarose gel. As control for specificity, the amplifiedPCR-products were sequenced by MWG-Biotech.

IMMUNOBLOT ANALYSIS: Western blot experiments were performed on 16.5%tricine-SDS-polyacrylamide gels. Proteins from human, mouse, and ratkidney and liver, as well as from human urine (50 ml for eachexperiment) were extracted according to the protocols published(Kulaksiz et al., (2003) Gut, in press; Kulaksiz et al., (2002) ProcNatl Acad Sci USA, 99:6796-6801; and Kulaksiz et al., (2002) Am JPathol, 161:655-664). Following electrophoresis, proteins weretransferred onto hydrophobic polyvinylidene fluoride-based membranes(Pall, Portsmouth, England) by semidry blotting. The membranes wereincubated overnight with hepcidin antibodies diluted 1:1000. Afterwashing in Tris-buffered saline containing 10 mM Tris-HCl (pH 8.0), 150mM NaCl, and 0.05% Tween 20, immunoreactive proteins were visualizedafter incubation with alkaline phosphatase-conjugated goat anti-rabbitantibody (diluted 1:50,000; Sigma) using nitro blue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate as chromogens (Sigma). Theimmunoreaction on the Western blot was specifically blocked afterpreincubation of the antibodies with the corresponding peptideimmunogens. Crossreactivity with the second goat anti-rabbit antibodywas excluded by appropriate controls (Kulaksiz et al., (2002) Proc NatlAcad Sci USA, 99:6796-6801; and Kulaksiz et al., (2002) Am J Pathol,161:655-664).

IMMUNOCYTOCHEMICAL PROTOCOL: Tissues were fixed in 4% paraformaldehydeor in Bouin's fixative for 18 h at 4° C. and embedded in paraffin.Paraffin sections (4-5 μm) were immunostained for hepcidin (antibodiesEG(1)-HepN, EG(2)-HepN, EG(1)-HepC, and EG(2)-HepC, each diluted 1:2000)by the avidin-biotin-peroxidase complex (ABC) technique; the incubationsequences and the visualization of the antigen-antibody binding siteswere performed as detailed (Kulaksiz et al., (2003) Gut, in press;Kulaksiz et al., (2002) Proc Natl Acad Sci USA, 99:6796-6801; andKulaksiz et al., (2002) Am J Pathol, 161:655-664). In brief, thesections were incubated with the respective antibodies for 24 h at 4°C., followed by incubation with biotinylated anti-rabbit IgG (JacksonImmunoresearch, West Grove, Pa., USA) for 30 min diluted 1:200. Thesections were then incubated for 30 min with a preformed complex ofbiotin-peroxidase/streptavidin (Jackson Immunoresearch), diluted in PBS(final concentrations: biotin-peroxidase, 0.7 μg/ml; streptavidin, 5μg/ml). The antigen-antibody binding sites were detected by incubationof the sections in 0.7 mM diaminobenzidine hydrochloride/0.002% H₂O₂ in0.05 M Tris-HCl (pH 7.6).

SPECIFICITY CONTROLS: Method-dependent non-specificities were excludedby running controls as published (Kulaksiz et al., (2003) Gut, inpress). Antibody specificities were tested by preadsorption of theantibodies with homologous and heterologous antigenic peptides (6.25-100μg/ml of the antiserum) (Kulaksiz et al., (2002) Proc Natl Acad Sci USA,99:6796-6801; and Kulaksiz et al., (2002) Am J Pathol, 161:655-664).Preadsorption of the antibodies with homologous antigens atconcentrations as low as 6.25 μg/ml completely blocked immunostaining inthe kidney, while preadsorption of the antibodies with heterologousantigens at concentrations up to 100 μg/ml had no effect onimmunostaining.

HEPCIDIN ELISA COMPETITIVE BINDING ASSAY: Serum and urine samples wereobtained from 22 individuals (11 women, 11 men, aged 23-59, mean 39 yrs)and serum samples were obtained from 22 patients with renalinsufficiency undergoing chronic hemodialysis (11 women, 11 men, aged25-77 years, mean 48 years). All patients with chronic renalinsufficiency were treated 2-3 times a week with 3,000 IE recombinanthuman erythropoietin (EPO). During sample collection it was taken carethat the healthy volunteers and patients had no infection and nobleeding. 10 ml blood samples were withdrawn into serum-tubes and 10 mlurine samples were collected in urine-tubes, centrifuged at 2,500×g for10 min at 4° C. Determinations were performed in duplicate using96-well-microtiter plates as described (8). In brief, microtiter plateswere coated with 200 μl/well rabbit anti-hepcidin antibody EG(2)-HepNdiluted 1:4000. 50 μl standards containing various amounts of syntheticpeptides (0, 20, 100, 500, and 1000 ng/ml) or human serum and urinesamples and 150 μl N-terminally biotinylated hepcidin-(28-47) (PeptideSpecialty Laboratories GnibH, Heidelberg, Germany) (2 ng/well) wereadded to each well and incubated for 1 hour at RT. After washing withTBST (TBS with 0.05% Tween 20), the biotinylated antigen-antibodycomplexes were detected by streptavidin-peroxidase enzyme (Dako,Hamburg, Germany) with the substrate tetramethylbenzidine (DRGInstruments GmbH, Marburg, Germany); the color reaction was stopped with1 M H₂SO₄ and the extinction of the solution was read at 450/630 nmwavelength.

STATISTICAL ANALYSIS: Data are illustrated as means±SEM. The statisticalanalysis was evaluated by Student's t-test. The differences wereconsidered significant at P<0.05.

Results

EXPRESSION OF HEPCIDIN IN THE MAMMALIAN KIDNEY: RT-PCR analysis revealedclear expression of hepcidin not only in the liver (positive control,see Kulaksiz et al., (2003) Gut, in press), but also in human, rat andmouse kidney (FIG. 8). A 192-bp expected PCR product for man, a 193-bpproduct for mouse, 201-bp product for rat were detected in the liver(data not shown) and kidney of these species. The sequence analysisrevealed that the PCR-generated products have a complete homology withthe cDNAs of the corresponding peptides.

At the translational level, the presence of hepcidin was confirmed byWestern blotting studies with region-specific antibodies (FIG. 8).Antisera directed against the C and N terminus of the hepcidin precursormolecule coincidentally identified an immunoreactive band of ˜9.5 kDa inextracts of human, rat and mouse kidney.

CELLULAR LOCALIZATION OF HEPCIDIN: Immunohistochemical studies withregion-specific hepcidin antisera consistently localized hepcidin to thedistal tubulus system of the human, mouse and rat kidney (FIGS. 9-13).The proximal renal tubuli, collecting tubuli, and the glomerulicompletely lacked hepcidin immunoreactivity. The immunoreactive ditaltubuli were confined to the renal cortex and outer renal medulla, theinner renal medulla showed no immunostaining for hepcidin (FIGS. 9, 10).Notably, distinct intercellular differences exist between the hepcidinpositive tubuli cells: while the majority of tubuli cells was stronglypositive for hepcidin, some of them showed only a faint immunoreactivityor were totally unreactive for hepcidin (FIG. 12). Conspicuously, in allsections investigated, the hepcidin antisera revealed a granularimmunoreactivity pattern in the cytoplasm of

epithelial cells lining the distal tubuli (FIGS. 9, 10). In sometissues, hepcidin-positive cells showed strong immunoreactivityconcentrated at the apical pole of the secretory cells (FIGS. 11, 13),and no immunoreactivity was found at the basolateral membrane domain ofthe respective cells.

DETECTION OF HEPCIDIN PROPEPTIDE IN SERUM AND URINE: A stable hepcidinELISA assay with high reproducibility and sensitivity was developed withthe specific N-terminal hepcidin antibody EG(2)-HepN (Kulaksiz et al.,(2003) Gut, in press). As seen in FIG. 14, the ELISA revealed thatpro-hepcidin is present in human serum. Pro25 hepcidin was measured inthe range from 68.5 to 139.2 ng/ml (mean±SE; 104.2±19.5 ng/ml) in theserum of healthy subjects. The prohepcidin concentrations varied from63.9 to 327.3 ng/ml (mean±SE; 156.8±61.9 ng/ml) in the serum of patientssuffering from chronic renal insufficiency and were significantlyincreased compared with that in control group.

Using the sensitive hepcidin ELISA, pro-hepcidin was detected in humanurine from the control group in the range from 13.9 to 456.0 ng/ml(mean±SE; 180.1±94.8 ng/ml). The presence of prohepcidin in human urinewas also confirmed by Western blotting analysis. Hepcidin antiseraidentified in extracts of human urine a single hepcidin immunoreactiveband of ˜9.5 kDa molecular mass that comigrated exactly with theimmunoreactive hepcidin in kidney tissues (FIG. 8)

Discussion

The new hormone hepcidin is an antimicrobial peptide and a centralregulator of iron homeostasis (Park et al., (2001) J Bid Chem,276:7806-7810; Krause et al., (2000) FEBS Lett, 480:147-150; Pigeon etal., (2001) J Biol Chem, 276:7811-7819; Nicolas et al., (2001) Proc NatlAcad Sci USA, 98:8780-8785; and Nicolas et al., (2002) Proc Natl AcadSci USA, 99:4596-4601). In previous studies, it was shown that the liveris the main source of hepcidin (Park et al., CH (2001) J Bid Chem,276:7806-7810; and Kulaksiz et al., (2003) Gut, in press). Althoughhepcidin was originally isolated from human urine (Park et al., (2001)and hemofiltrate (Krause et al., (2000)), no expression of thisregulatory peptide was detected in the kidney (Pigeon et al., (2001).

Using the appropriate primer specifications and combinationssuccessfully employed in the liver (Kulaksiz et al., (2003) Gut, inpress; and Gehrke et al., (2003) Blood, 102:371-376), the present RT-PCRanalysis clearly revealed that hepcidin is not only expressed in theliver, but also in the kidney of three mammalian species—man, rat andmouse. Sequencing analysis revealed the specificity of the generatedPCR-products.

To verify the presence of the translated hepcidin peptide in the kidney,Applicants raised a bulk of region-specific antisera against hepcidinand used them in Western blotting analyses and immunohistochemistry.Western blot analysis confirmed expression of hepcidin in the kidney.Four different antisera recognizing different epitopes in the hepcidinprecursor molecule concurrently identified an immunoreactive peptide of˜9.5 kDa in the kidney of three different species, which corresponds tothe molecular mass of hepcidin prohormone deduced from the respectivecDNA sequence (Pigeon et al., (2001)). The apparent molecular mass ofthis immunoreactive peptide is also in accordance with the molecularmass of hepcidin prohormone detected in the liver (Kulaksiz et al.,(2003) Gut, in press). Applicants' findings unequivocaly demonstratedthat hepcidin is not liver specific, since it is also present in thekidney.

Immunocytochemical investigations with four region-specific hepcidinantisera revealed that in human, mouse, and rat kidney, hepcidin isspecifically localized to the tubule system of the renal cortex andouter medulla. These immunoreactive tubules were identified as distalrenal tubuli by their typical morphological feature detected in lightmicroscopy. Coincident staining by different region-specific antibodiesnot only in the human, but also in mouse and rat kidney points to distaltubuli being the source of renal hepcidin. No immunoreactivity forhepcidin was detected in the proximal renal tubuli, collecting tubuli,and the glomeruli or the renal inner medulla.

In the renal cortex and outer medulla, hepcidin immunoreactivity wasconfined to epithelial secretory cells of the distal tubuli. Remarkably,all hepcidin antisera produced a granular immunoreactivity patternassuming localization of the peptide in small secretory vesicles orlysosomes of the respective cells that have been already identified inthese cells by electron microscopy (van Katachalan M A, Kritz W:Pathology of the kidney. Edited by J C Jennette, J L Oldson, M MSchwarz, S G Silver: Philadelphia, Heptinstall's, 1998, pp 3-66).Notably, distinct intercellular differences exist between the epithelialcells even of the same tubulus with respect to the density of hepcidinimmunoreactivity that may reflect intercellular differences inexpression or secretion of hepcidin. Conspicuously, in some tubuliimmunoreactivity for hepcidin was localized in the whole cytoplasm ofthe epithelial cells whereas in other tubuli strong hepcidinimmunoreactivity was concentrated at the apical pole of the secretorycells. This peculiar distribution pattern of hepcidin at the cellularlevel may assume a luminally directed release of hepcidin. Applicantsdid not detect hepcidin expression at the basolateral membrane domain ofrenal tubuli cells. This suggests that renal hepcidin is not releasedinto the blood by secretory cells lining the tubuli.

It is widely believed that control of iron homeostasis of the body ismainly dependent on tight regulation of iron uptake from the diet in theproximal small intestine. However, recent studies have shown that thekidney plays an important role in iron homeostasis (Wareing et al.,(2003) Am J Physiol Renal Physiol, Epub ahead of print; Ferguson et al.,(2003) Kidney mt1 64:1755-1764; and Gunshin et al., (1997) Nature,388:482-488). Wareing and co-workers could convincingly demonstrate thata metabolically significant amount of iron is filtered at the glomerulusand only 0.8-1.5% of the filtered iron is actually excreted in the urine(Wareing et al., (2000) J Physiol, 524.2:581-586). Thus, there must be avery effective pathway and a strong regulation for reabsorption of ironalong the renal tubules. Indeed, Ferguson and co-workers could localizethe divalent metal transporter 1 (DMT-1) in the tubule system of thekidney (Ferguson et al., (2001) Am J Physiol Renal Physiol,280:F803-F814). This protein is proposed to be the major pathway foruptake of dietary iron by the gastrointestinal tract (Gunshin et al.,(1997)). Notably, DMT-1 expression has been shown to be highest at theapical membrane domaine of the tubuli cells of renal cortex and outermedulla, where Applicants also found hepcidin. Furthermore, recentstudies demonstrated that altered dietary iron intake strongly modulatesrenal DMT-1 expression (Wareing et al., (2003)). Based on these findingsand data showing that hepcidin expression inversely correlates with theexpression of duodenal DMT-1 (Frazer et al., (2002) Gastroenterology,123:835-844), Applicants suggest a regulatory role for hepcidin in renaliron transport.

The potential release of hepcidin into the urine was substantiated byWestern blot studies. The region-specific hepcidin-antiseracoincidentally identified a strongly labeled band of correct molecularmass (Kulaksiz et al., (2003) Gut, in press) that co-migrated exactlywith the immunoreactive prohepcidin as in kidney tissue extracts. Thesefindings clearly show that prohepcidin is synthesized by secretorydistal tubuli cells and released luminally into the urine where itapparently escapes tubular proteolysis and recycling. To measure theprohepcidin concentration in human urine, a sensitive ELISA with adetection sensitivity of 3.95 ng/well was developed. ELISA analyses withthe hepcidin antiserum EG(2)-HepN, which was already successfully usedin ELISA experiments (Kulaksiz et al., (2003) Gut, in press) revealed ahigh concentration of prohepcidin in the range from 13.9 to 456.0 ng/ml(mean±SE; 180.1±94.8 ng/ml) in urine of healthy subjects. Thisconcentration is considerably higher than the prohepcidin concentrationof the same persons in the circulation (68.5 to 139.2 ng/ml; mean±SE,104.2±19.5 ng/rnl). Notably, no correlation was found between thecirculating pro-hepcidin and the serum iron or ferritin levels(ICulaksiz et al., (2003) Gut, in press). Likewise, no correlation wasdetected between urinary prohepcidin and serum iron or ferritin levels(data not shown), that are supposed to regulate the expression of liverhepcidin (Pigeon et al., (2001) J Biol Chem, 276:7811-7819; Nemeth etal., (2002 Blood, 101:2461-2463; and Ganz T, (2003) Blood, 102:783-788).Therefore, Applicants suggest that the regulation of renal/urinarypro-hepcidin is not directly influenced by serum iron or ferritin.

The evaluation of pro-hepcidin regulation in renal insufficient patientsundergoing chronic hemodialysis revealed that concentrations ofpro-hepcidin in the serum of these patients were significantly increasedfrom 104.2 ng/ml in healthy subjects to 156.8 ng/ml. Enhanced levels ofpro-hepcidin in dialysis patients suggests that the kidneys are not onlyinvolved in the synthesis of hepcidin, but they may also be involved inthe metabolism and/or elimination of the circulating peptide.Interestingly, in a current study the kidney hormone erythropoietin hasbeen shown to down-regulate the liver hepcidin gene expression (Nicolas(2002) Blood Cells, Molecules, and Diseases, 29:327-335). Thus, anotherexplanation for the enhanced pro-hepcidin concentrations in dialysispatients could be the relative deficiency of erythropoietin, which isencountered regularly in terminal renal insufficiency (Eckardt K U,(2000) Clin Nephrol, 53:S2-8; and Santoro A: (2002) Rev din Exp Hematol,Suppi 1:12-20). However, Applicants report enhanced levels ofpro-hepcidin measured in patients with chronic renal insufficiency,although they were treated with the hepcidin inhibitory hormoneerythropoietin, supporting the renal filtration of hepcidin. Oneembodiment of the present invention provides that urinary hepcidinoriginates partly from the kidney and partly from the liver; therefore,it has to be noticed that the measured urinary pro-hepcidin is a totalof released renal peptide and the eliminated circulating peptide.

Summing up, recent studies demonstrate that the kidney plays animportant role in iron homeostasis (Wareing et al., (2003) Am J PhysiolRenal Physiol, Epub ahead of print; Ferguson et al., (2003) Kidney Int,64:1755-1764; Gunshin et al., (1997) Nature, 388:482-488; Wareing etal., (2000) J Physiol, 524.2:581-586 and Ferguson et al., (2001) Am JPhysiol Renal Physiol, 280:F803-F814), however, no data exists about theregulatory mechanism of the renal iron transport. In this respect,Applicants localized hepcidin for the first time in the kidney of threemammalian species. Applicants' findings indicate that hepcidin is notliver-specific. Beside the elimination of serum hepcidin in the kidney,the peptide is also produced as an intrinsic hormone in the secretorydistal tubuli cells of the kidney and released luminally into the urine,implicating a regulatory role for hepcidin in the kidney and/or urinarytract. Further studies should analyze the regulatory mechanism ofhepcidin in the renal tubule system.

Example 3 Expression of Hepcidin in the Human Pancreas

Pancreatic tissues used in the present study were obtained after theWhipple operation in patients suffering from pancreatic cancer. Usingthe appropriate primer specifications and combinations successfullyemployed in the liver and kidney, the present RT-PCR analysis revealedthat hepcidin is not only expressed in the liver and kidney, but also inthe human pancreas. Sequencing analysis revealed the specificity of thegenerated PCR products.

Western blot analyses with specific antibodies confirmed the expressionof hepcidin in the pancreas at the translational level. Using the sameantibodies, hepcidin was localized in the pancreas byimmunohistochemistry. Paraffin sections revealed that hepcidinimmunoreactivity is localized in the endocrine pancreas; noimmunoreactivity was found in the exocrine pancreas.

INDUSTRIAL APPLICABILITY

The invention has applications in connection with diagnosing a diseasecondition characterized by non-physiological levels of hepcidin protein,including prohepcidin and fragments thereof.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

All patent and non-patent publications cited in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains. All these publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated herein by reference.

1. A method of detecting hepcidin comprising the steps of: (a) obtaininga tissue or fluid sample from a subject; and (b) contacting the samplewith an antibody or fragment thereof that specifically binds to one ormore carboxy terminal epitopes contained within amino acids 70 to 84 ofSEQ ID NO: 2; wherein the tissue or fluid sample is selected from thegroup consisting of a kidney sample, a liver sample, and a urine sample,and wherein the method of detecting hepcidin is selected from the groupconsisting of Western blot, immunodot, immunohistochemistry, andimmunofluorescence.
 2. The method of claim 1, wherein said hepcidin ishepcidin, prohepcidin or fragments thereof.
 3. The method of claim 1,wherein said hepcidin is prohepcidin.